Isolated human transporter proteins, nucleic acid molecules encoding human transporter proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the transporter peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the transporter peptides, and methods of identifying modulators of the transporter peptides.

RELATED APPLICATIONS

[0001] The present application claims priority to provisionalapplication U.S. Serial No. 760/254,553, filed Dec. 12, 2004 (Atty.Docket CL001014-PROV), and application U.S. Ser. No. 09/739,457, filedDec. 19, 2000 (Atty. Docket CL001014).

FIELD OF THE INVENTION

[0002] The present invention is in the field of transporter proteinsthat are related to the sugar transporter subfamily, recombinant DNAmolecules, and protein production. The present invention specificallyprovides novel peptides and proteins that effect ligand transport andnucleic acid molecules encoding such peptide and protein molecules, allof which are useful in the development of human therapeutics anddiagnostic compositions and methods.

BACKGROUND OF THE INVENTION

[0003] Transporters

[0004] Transporter proteins regulate many different functions of a cell,including cell proliferation, differentiation, and signaling processes,by regulating the flow of molecules such as ions and macromolecules,into and out of cells. Transporters are found in the plasma membranes ofvirtually every cell in eukaryotic organisms. Transporters mediate avariety of cellular functions including regulation of membranepotentials and absorption and secretion of molecules and ion across cellmembranes. When present in intracellular membranes of the Golgiapparatus and endocytic vesicles, transporters, such as chloridechannels, also regulate organelle pH. For a review, see Greger, R.(1988) Annu. Rev. Physiol. 50:111-122.

[0005] Transporters are generally classified by structure and the typeof mode of action. In addition, transporters are sometimes classified bythe molecule type that is transported, for example, sugar transporters,chlorine channels, potassium channels, etc. There may be many classes ofchannels for transporting a single type of molecule (a detailed reviewof channel types can be found at Alexander, S. P. H. and J. A. Peters:Receptor and transporter nomenclature supplement. Trends Pharmacol.Sci., Elsevier, pp. 65-68 (1997) andhttp://www-biology.ucsd.edu/˜msaier/transport/titlepage2.html.

[0006] The following general classification scheme is known in the artand is followed in the present discoveries.

[0007] Channel-type transporters. Transmembrane channel proteins of thisclass are ubiquitously found in the membranes of all types of organismsfrom bacteria to higher eukaryotes. Transport systems of this typecatalyze facilitated diffusion (by an energy-independent process) bypassage through a transmembrane aqueous pore or channel without evidencefor a carrier-mediated mechanism. These channel proteins usually consistlargely of a-helical spanners, although b-strands may also be presentand may even comprise the channel. However, outer membrane porin-typechannel proteins are excluded from this class and are instead includedin class 9.

[0008] Carrier-type transporters. Transport systems are included in thisclass if they utilize a carrier-mediated process to catalyze uniport (asingle species is transported by facilitated diffusion), antiport (twoor more species are transported in opposite directions in a tightlycoupled process, not coupled to a direct form of energy other thanchemiosmotic energy) and/or symport (two or more species are transportedtogether in the same direction in a tightly coupled process, not coupledto a direct form of energy other than chemiosmotic energy).

[0009] Pyrophosphate bond hydrolysis-driven active transporters.Transport systems are included in this class if they hydrolyzepyrophosphate or the terminal pyrophosphate bond in ATP or anothernucleoside triphosphate to drive the active uptake and/or extrusion of asolute or solutes. The transport protein may or may not be transientlyphosphorylated, but the substrate is not phosphorylated.

[0010] PEP-dependent, phosphoryl transfer-driven group translocators.Transport systems of the bacterial phosphoenolpyruvate:sugarphosphotransferase system are included in this class. The product of thereaction, derived from extracellular sugar, is a cytoplasmicsugar-phosphate.

[0011] Decarboxylation-driven active transporters. Transport systemsthat drive solute (e.g., ion) uptake or extrusion by decarboxylation ofa cytoplasmic substrate are included in this class.

[0012] Oxidoreduction-driven active transporters. Transport systems thatdrive transport of a solute (e.g., an ion) energized by the flow ofelectrons from a reduced substrate to an oxidized substrate are includedin this class. Light-driven active transporters. Transport systems thatutilize light energy to drive transport of a solute (e.g., an ion) areincluded in this class.

[0013] Mechanically-driven active transporters. Transport systems areincluded in this class if they drive movement of a cell or organelle byallowing the flow of ions (or other solutes) through the membrane downtheir electrochemical gradients.

[0014] Outer-membrane porins (of b-structure). These proteins formtransmembrane pores or channels that usually allow the energyindependent passage of solutes across a membrane. The transmembraneportions of these proteins consist exclusively of b-strands that form ab-barrel. These porin-type proteins are found in the outer membranes ofGram-negative bacteria, mitochondria and eukaryotic plastids.

[0015] Methyltransferase-driven active transporters. A singlecharacterized protein currently falls into this category, theNa+-transporting methyltetrahydromethanopterin:coenzyme Mmethyltransferase.

[0016] Non-ribosome-synthesized channel-forming peptides or peptide-likemolecules. These molecules, usually chains of L- and D-amino acids aswell as other small molecular building blocks such as lactate, formoligomeric transmembrane ion channels. Voltage may induce channelformation by promoting assembly of the transmembrane channel. Thesepeptides are often made by bacteria and fungi as agents of biologicalwarfare.

[0017] Non-Proteinaceous Transport Complexes. Ion conducting substancesin biological membranes that do not consist of or are not derived fromproteins or peptides fall into this category.

[0018] Functionally characterized transporters for which sequence dataare lacking. Transporters of particular physiological significance willbe included in this category even though a family assignment cannot bemade.

[0019] Putative transporters in which no family member is an establishedtransporter. Putative transport protein families are grouped under thisnumber and will either be classified elsewhere when the transportfunction of a member becomes established, or will be eliminated from theTC classification system if the proposed transport function isdisproven. These families include a member or members for which atransport function has been suggested, but evidence for such a functionis not yet compelling.

[0020] Auxiliary transport proteins. Proteins that in some wayfacilitate transport across one or more biological membranes but do notthemselves participate directly in transport are included in this class.These proteins always function in conjunction with one or more transportproteins. They may provide a function connected with energy coupling totransport, play a structural role in complex formation or serve aregulatory function.

[0021] Transporters of unknown classification. Transport proteinfamilies of unknown classification are grouped under this number andwill be classified elsewhere when the transport process and energycoupling mechanism are characterized. These families include at leastone member for which a transport function has been established, buteither the mode of transport or the energy coupling mechanism is notknown.

[0022] Ion Channels

[0023] An important type of transporter is the ion channel. Ion channelsregulate many different cell proliferation, differentiation, andsignaling processes by regulating the flow of ions into and out ofcells. Ion channels are found in the plasma membranes of virtually everycell in eukaryotic organisms. Ion channels mediate a variety of cellularfunctions including regulation of membrane potentials and absorption andsecretion of ion across epithelial membranes. When present inintracellular membranes of the Golgi apparatus and endocytic vesicles,ion channels, such as chloride channels, also regulate organelle pH. Fora review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

[0024] Ion channels are generally classified by structure and the typeof mode of action. For example, extracellular ligand gated channels(ELGs) are comprised of five polypeptide subunits, with each subunithaving 4 membrane spanning domains, and are activated by the binding ofan extracellular ligand to the channel. In addition, channels aresometimes classified by the ion type that is transported, for example,chlorine channels, potassium channels, etc. There may be many classes ofchannels for transporting a single type of ion (a detailed review ofchannel types can be found at Alexander, S. P. H. and J. A. Peters(1997). Receptor and ion channel nomenclature supplement. TrendsPharmacol. Sci., Elsevier, pp. 65-68 andhttp://www-biology.ucsd.edu/˜msaier/transport/toc.html.

[0025] There are many types of ion channels based on structure. Forexample, many ion channels fall within one of the following groups:extracellular ligand-gated channels (ELG), intracellular ligand-gatedchannels (ILG), inward rectifying channels (INR), intercellular (gapjunction) channels, and voltage gated channels (VIC). There areadditionally recognized other channel families based on ion-typetransported, cellular location and drug sensitivity. Detailedinformation on each of these, their activity, ligand type, ion type,disease association, drugability, and other information pertinent to thepresent invention, is well known in the art.

[0026] Extracellular ligand-gated channels, ELGs, are generallycomprised of five polypeptide subunits, Unwin, N. (1993), Cell 72:31-41; Unwin, N. (1995), Nature 373: 3743; Hucho, F., et al., (1996) J.Neurochem. 66: 1781-1792; Hucho, F., et al., (1996) Eur. J. Biochem.239: 539-557; Alexander, S. P. H. and J. A. Peters (1997), TrendsPharmacol. Sci., Elsevier, pp. 4-6; 36-40; 4244; and Xue, H. (1998) J.Mol. Evol. 47: 323-333. Each subunit has 4 membrane spanning regions:this serves as a means of identifying other members of the ELG family ofproteins. ELG bind a ligand and in response modulate the flow of ions.Examples of ELG include most members of the neurotransmitter-receptorfamily of proteins, e.g., GABAI receptors. Other members of this familyof ion channels include glycine receptors, ryandyne receptors, andligand gated calcium channels.

[0027] The Voltage-gated Ion Channel (VIC) Superfamily

[0028] Proteins of the VIC family are ion-selective channel proteinsfound in a wide range of bacteria, archaea and eukaryotes Hille, B.(1992), Chapter 9: Structure of channel proteins; Chapter 20: Evolutionand diversity. In: Ionic Channels of Excitable Membranes, 2nd Ed.,Sinaur Assoc. Inc., Pubs., Sunderland, Mass.; Sigworth, F. J. (1993),Quart. Rev. Biophys. 27: 140; Salkoff, L. and T. Jegla (1995), Neuron15: 489492; Alexander, S. P. H. et al., (1997), Trends Pharmacol. Sci.,Elsevier, pp. 76-84; Jan, L. Y. et al., (1997), Annu. Rev. Neurosci. 20:91-123; Doyle, D. A, et al., (1998) Science 280: 69-77; Terlau, H. andW. Stühmer (1998), Naturwissenschaften 85: 437-444. They are often homo-or heterooligomeric structures with several dissimilar subunits (e.g.,a1-a2-d-b Ca²⁺ channels, ab₁b₂ Na⁺ channels or (a)₄-b K⁺ channels), butthe channel and the primary receptor is usually associated with the a(or a1) subunit. Functionally characterized members are specific for K⁺,Na⁺ or Ca²⁺. The K⁺ channels usually consist of homotetramericstructures with each a-subunit possessing six transmembrane spanners(TMSs). The al and a subunits of the Ca²⁺ and Na⁺ channels,respectively, are about four times as large and possess 4 units, eachwith 6 TMSs separated by a hydrophilic loop, for a total of 24 TMSs.These large channel proteins form heterotetra-unit structures equivalentto the homotetrameric structures of most K⁺ channels. All four units ofthe Ca²⁺ and Na⁺ channels are homologous to the single unit in thehomotetrameric K⁺ channels. Ion flux via the eukaryotic channels isgenerally controlled by the transmembrane electrical potential (hencethe designation, voltage-sensitive) although some are controlled byligand or receptor binding.

[0029] Several putative K⁺-selective channel proteins of the VIC familyhave been identified in prokaryotes. The structure of one of them, theKcsA K⁺ channel of Streptomyces lividans, has been solved to 3.2 Åresolution. The protein possesses four identical subunits, each with twotransmembrane helices, arranged in the shape of an inverted teepee orcone. The cone cradles the “selectivity filter” P domain in its outerend. The narrow selectivity filter is only 12 Å long, whereas theremainder of the channel is wider and lined with hydrophobic residues. Alarge water-filled cavity and helix dipoles stabilize K⁺ in the pore.The selectivity filter has two bound K⁺ ions about 7.5 Å apart from eachother. Ion conduction is proposed to result from a balance ofelectrostatic attractive and repulsive forces.

[0030] In eukaryotes, each VIC family channel type has several subtypesbased on pharmacological and electrophysiological data. Thus, there arefive types of Ca²⁺ channels (L, N, P, Q and T). There are at least tentypes of K⁺ channels, each responding in different ways to differentstimuli: voltage-sensitive [Ka, Kv, Kvr, Kvs and Ksr], Ca²⁺-sensitive[BK_(ca), IK_(Ca) and SK_(ca)] and receptor-coupled [K_(M) and K_(Ach)].There are at least six types of Na⁺ channels (I, II, III, μ1, H1 andPN3). Tetrameric channels from both prokaryotic and eukaryotic organismsare known in which each a-subunit possesses 2 TMSs rather than 6, andthese two TMSs are homologous to TMSs 5 and 6 of the six TMS unit foundin the voltage-sensitive channel proteins. KcsA of S. lividans is anexample of such a 2 TMS channel protein. These channels may include theK_(Na) (Na⁺-activated) and K_(Vol) (cell volume-sensitive) K⁺ channels,as well as distantly related channels such as the Tokl K⁺ channel ofyeast, the TWIK-1 inward rectifier K⁺ channel of the mouse and theTREK-1 K⁺ channel of the mouse. Because of insufficient sequencesimilarity with proteins of the VIC family, inward rectifier K⁺ IRKchannels (ATP-regulated; G-protein-activated) which possess a P domainand two flanking TMSs are placed in a distinct family. However,substantial sequence similarity in the P region suggests that they arehomologous. The b, g and d subunits of VIC family members, when present,frequently play regulatory roles in channel activation/deactivation.

[0031] The Epithelial Na⁺ Channel (ENaC) Family

[0032] The ENaC family consists of over twenty-four sequenced proteins(Canessa, C. M., et al., (1994), Nature 367: 463-467, Le, T. and M. H.Saier, Jr. (1996), Mol. Membr. Biol. 13: 149-157; Garty, H. and L. G.Palmer (1997), Physiol. Rev. 77: 359-396; Waldmann, R., et al., (1997),Nature 386: 173-177; Darboux, I., et al., (1998), J. Biol. Chem. 273:9424-9429; Firsov, D., et al., (1998), EMBO J. 17: 344-352; Horisberger,J.-D. (1998). Curr. Opin. Struc. Biol. 10: 443-449). All are fromanimals with no recognizable homologues in other eukaryotes or bacteria.The vertebrate ENaC proteins from epithelial cells cluster tightlytogether on the phylogenetic tree: voltage-insensitive ENaC homologuesare also found in the brain. Eleven sequenced C. elegans proteins,including the degenerins, are distantly related to the vertebrateproteins as well as to each other. At least some of these proteins formpart of a mechano-transducing complex for touch sensitivity. Thehomologous Helix aspersa (FMRF-amide)-activated Na⁺ channel is the firstpeptide neurotransmitter-gated ionotropic receptor to be sequenced.

[0033] Protein members of this family all exhibit the same apparenttopology, each with N- and C-termini on the inside of the cell, twoamphipathic transmembrane spanning segments, and a large extracellularloop. The extracellular domains contain numerous highly conservedcysteine residues. They are proposed to serve a receptor function.

[0034] Mammalian ENaC is important for the maintenance of Na⁺ balanceand the regulation of blood pressure. Three homologous ENaC subunits,alpha, beta, and gamma, have been shown to assemble to form the highlyNa⁺-selective channel. The stoichiometry of the three subunits isalpha₂, beta1, gammal in a heterotetrameric architecture.

[0035] The Glutamate-gated Ion Channel (GIC) Family of NeurotransmitterReceptors

[0036] Members of the GIC family are heteropentameric complexes in whicheach of the 5 subunits is of 800-1000 amino acyl residues in length(Nakanishi, N., et al, (1990), Neuron 5: 569-581; Unwin, N. (1993), Cell72: 3141; Alexander, S. P. H. and J. A. Peters (1997) Trends Pharmacol.Sci., Elsevier, pp. 36-40). These subunits may span the membrane threeor five times as putative a-helices with the N-termini (theglutamate-binding domains) localized extracellularly and the C-terminilocalized cytoplasmically. They may be distantly related to theligand-gated ion channels, and if so, they may possess substantialb-structure in their transmembrane regions. However, homology betweenthese two families cannot be established on the basis of sequencecomparisons alone. The subunits fall into six subfamilies: a, b, g, d, eand z.

[0037] The GIC channels are divided into three types: (1)a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-, (2) kainate-and (3) N-methyl-D-aspartate (NMDA)-selective glutamate receptors.Subunits of the AMPA and kainate classes exhibit 35-40% identity witheach other while subunits of the NMDA receptors exhibit 22-24% identitywith the former subunits. They possess large N-terminal, extracellularglutamate-binding domains that are homologous to the periplasmicglutamine and glutamate receptors of ABC-type uptake permeases ofGram-negative bacteria. All known members of the GIC family are fromanimals. The different channel (receptor) types exhibit distinct ionselectivities and conductance properties. The NMDA-selective largeconductance channels are highly permeable to monovalent cations andCa²⁺. The AMPA- and kainate-selective ion channels are permeableprimarily to monovalent cations with only low permeability to Ca²⁺.

[0038] The Chloride Channel (ClC) Family

[0039] The ClC family is a large family consisting of dozens ofsequenced proteins derived from Gram-negative and Gram-positivebacteria, cyanobacteria, archaea, yeast, plants and animals (Steinmeyer,K., et al., (1991), Nature 354: 301-304; Uchida, S., et al., (1993), J.Biol. Chem. 268: 3821-3824; Huang, M.-E., et al., (1994), J. Mol. Biol.242: 595-598; Kawasaki, M., et al, (1994), Neuron 12: 597-604; Fisher,W. E., et al., (1995), Genomics. 29:598-606; and Foskett, J. K. (1998),Annu. Rev. Physiol. 60: 689-717). These proteins are essentiallyubiquitous, although they are riot encoded within genomes of Haemophilusinfluenzae, Mycoplasma genitalium, and Mycoplasma pneumoniae. Sequencedproteins vary in size from 395 amino acyl residues (M. jannaschii) to988 residues (man). Several organisms contain multiple ClC familyparalogues. For example, Synechocystis has two paralogues, one of 451residues in length and the other of 899 residues. Arabidopsis thalianahas at least four sequenced paralogues, (775-792 residues), humans alsohave at least five paralogues (820-988 residues), and C. elegans alsohas at least five (810-950 residues). There are nine known members inmammals, and mutations in three of the corresponding genes cause humandiseases. E. coli, Methanococcus jannaschii and Saccharomyces cerevisiaeonly have one ClC family member each. With the exception of the largerSynechocystis paralogue, all bacterial proteins are small (395-492residues) while all eukaryotic proteins are larger (687-988 residues).These proteins exhibit 10-12 putative transmembrane a-helical spanners(TMSs) and appear to be present in the membrane as homodimers. While onemember of the family, Torpedo ClC-O, has been reported to have twochannels, one per subunit, others are believed to have just one.

[0040] All functionally characterized members of the ClC familytransport chloride, some in a voltage-regulated process. These channelsserve a variety of physiological functions (cell volume regulation;membrane potential stabilization; signal transduction; transepithelialtransport, etc.). Different homologues in humans exhibit differing anionselectivities, i.e., ClC4 and ClC5 share a NO₃ ⁻>Cl⁻>Br⁻>I⁻ conductancesequence, while ClC3 has an I⁻>Cl⁻ selectivity. The ClC4 and ClC5channels and others exhibit outward rectifying currents with currentsonly at voltages more positive than +20 mV.

[0041] Animal Inward Rectifier K⁺ Channel (IRK-C) Family

[0042] IRK channels possess the “minimal channel-forming structure” withonly a P domain, characteristic of the channel proteins of the VICfamily, and two flanking transmembrane spanners (Shuck, M. E., et al.,(1994), J. Biol. Chem. 269: 24261-24270; Ashen, M. D., et al., (1995),Am. J. Physiol. 268: H506-H511; Salkoff, L. and T. Jegla (1995), Neuron15: 489-492; Aguilar-Bryan, L., et al., (1998), Physiol. Rev. 78:227-245; Ruknudin, A., et al., (1998), J. Biol. Chem. 273: 14165-14171).They may exist in the membrane as homo- or heterooligomers. They have agreater tendency to let K⁺ flow into the cell than out.Voltage-dependence may be regulated by external K⁺, by internal Mg²⁺, byinternal ATP and/or by G-proteins. The P domains of IRK channels exhibitlimited sequence similarity to those of the VIC family, but thissequence similarity is insufficient to establish homology. Inwardrectifiers play a role in setting cellular membrane potentials, and theclosing of these channels upon depolarization permits the occurrence oflong duration action potentials with a plateau phase. Inward rectifierslack the intrinsic voltage sensing helices found in VIC family channels.In a few cases, those of Kir1.1a and Kir6.2, for example, directinteraction with a member of the ABC superfamily has been proposed toconfer unique functional and regulatory properties to the heteromericcomplex, including sensitivity to ATP. The SUR1 sulfonylurea receptor(spQ09428) is the ABC protein that regulates the Kir6.2 channel inresponse to ATP, and CFTR may regulate Kir1.1a. Mutations in SUR1 arethe cause of familial persistent hyperinsulinemic hypoglycemia ininfancy (PHHI), an autosomal recessive disorder characterized byunregulated insulin secretion in the pancreas.

[0043] ATP-gated Cation Channel (ACC) Family

[0044] Members of the ACC family (also called P2X receptors) respond toATP, a functional neurotransmitter released by exocytosis from manytypes of neurons (North, R. A. (1996), Curr. Opin. Cell Biol. 8: 474483;Soto, F., M. Garcia-Guzman and W. Stühmer (1997), J. Membr. Biol. 160:91-100). They have been placed into seven groups (P2X₁-P2X₇) based ontheir pharmacological properties. These channels, which function atneuron-neuron and neuron-smooth muscle junctions, may play roles in thecontrol of blood pressure and pain sensation. They may also function inlymphocyte and platelet physiology. They are found only in animals.

[0045] The proteins of the ACC family are quite similar in sequence(>35% identity), but they possess 380-1000 amino acyl residues persubunit with variability in length localized primarily to the C-terminaldomains. They possess two transmembrane spanners, one about 30-50residues from their N-termini, the other near residues 320-340. Theextracellular receptor domains between these two spanners (of about 270residues) are well conserved with numerous conserved glycyl and cysteylresidues. The hydrophilic C-termini vary in length from 25 to 240residues. They resemble the topologically similar epithelial Na⁺ channel(ENaC) proteins in possessing (a) N- and C-termini localizedintracellularly, (b) two putative transmembrane spanners, (c) a largeextracellular loop domain, and (d) many conserved extracellular cysteylresidues. ACC family members are, however, not demonstrably homologouswith them. ACC channels are probably hetero- or homomultimers andtransport small monovalent cations (Me⁺). Some also transport Ca²⁺; afew also transport small metabolites.

[0046] The Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca²⁺ Channel(RIR-CaC) Family

[0047] Ryanodine (Ry)-sensitive and inositol 1,4,5-triphosphate(IP3)-sensitive Ca²⁺-release channels function in the release of Ca²⁺from intracellular storage sites in animal cells and thereby regulatevarious Ca²⁺-dependent physiological processes (Hasan, G. et al., (1992)Development 116: 967-975; Michikawa, T., et al., (1994), J. Biol. Chem.269: 9184-9189; Tunwell, R. E. A., (1996), Biochem. J. 318: 477487; Lee,A. G. (1996) Biomembranes, Vol. 6, Transmembrane Receptors and Channels(A. G. Lee, ed.), JAI Press, Denver, Colo., pp 291-326; Mikoshiba, K.,et al., (1996) J. Biochem. Biomem. 6: 273-289). Ry receptors occurprimarily in muscle cell sarcoplasmic reticular (SR) membranes, and IP3receptors occur primarily in brain cell endoplasmic reticular (ER)membranes where they effect release of Ca²⁺ into the cytoplasm uponactivation (opening) of the channel.

[0048] The Ry receptors are activated as a result of the activity ofdihydropyridine-sensitive Ca²⁺ channels. The latter are members of thevoltage-sensitive ion channel (VIC) family. Dihydropyridine-sensitivechannels are present in the T-tubular systems of muscle tissues.

[0049] Ry receptors are homotetrameric complexes with each subunitexhibiting a molecular size of over 500,000 daltons (about 5,000 aminoacyl residues). They possess C-terminal domains with six putativetransmembrane a -helical spanners (TMSs). Putative pore-formingsequences occur between the fifth and sixth TMSs as suggested formembers of the VIC family. The large N-terminal hydrophilic domains andthe small C-terminal hydrophilic domains are localized to the cytoplasm.Low resolution 3-dimensional structural data are available. Mammalspossess at least three isoforms that probably arose by gene duplicationand divergence before divergence of the mammalian species. Homologuesare present in humans and Caenorabditis elegans.

[0050] IP₃ receptors resemble Ry receptors in many respects. (1) Theyare homotetrameric complexes with each subunit exhibiting a molecularsize of over 300,000 daltons (about 2,700 amino acyl residues). (2) Theypossess C-terminal channel domains that are homologous to those of theRy receptors. (3) The channel domains possess six putative TMSs and aputative channel lining region between TMSs 5 and 6. (4) Both the largeN-terminal domains and the smaller C-terminal tails face the cytoplasm.(5) They possess covalently linked carbohydrate on extracytoplasmicloops of the channel domains. (6) They have three currently recognizedisoforms (types 1, 2, and 3) in mammals which are subject todifferential regulation and have different tissue distributions.

[0051] IP₃ receptors possess three domains: N-terminal IP₃-bindingdomains, central coupling or regulatory domains and C-terminal channeldomains. Channels are activated by IP₃ binding, and like the Ryreceptors, the activities of the IP₃ receptor channels are regulated byphosphorylation of the regulatory domains, catalyzed by various proteinkinases. They predominate in the endoplasmic reticular membranes ofvarious cell types in the brain but have also been found in the plasmamembranes of some nerve cells derived from a variety of tissues.

[0052] The channel domains of the Ry and IP₃ receptors comprise acoherent family that in spite of apparent structural similarities, donot show appreciable sequence similarity of the proteins of the VICfamily. The Ry receptors and the IP₃ receptors cluster separately on theRIR-CaC family tree. They both have homologues in Drosophila. Based onthe phylogenetic tree for the family, the family probably evolved in thefollowing sequence: (1) A gene duplication event occurred that gave riseto Ry and IP₃ receptors in invertebrates. (2) Vertebrates evolved frominvertebrates. (3) The three isoforms of each receptor arose as a resultof two distinct gene duplication events. (4) These isoforms weretransmitted to mammals before divergence of the mammalian species.

[0053] The Organellar Chloride Channel (O-ClC) Family

[0054] Proteins of the O-ClC family are voltage-sensitive chloridechannels found in intracellular membranes but not the plasma membranesof animal cells (Landry, D, et al., (1993), J. Biol. Chem. 268:14948-14955; Valenzuela, Set al., (1997), J. Biol. Chem. 272:12575-12582; and Duncan, R. R., et al., (1997), J. Biol. Chem. 272:23880-23886).

[0055] They are found in human nuclear membranes, and the bovine proteintargets to the microsomes, but not the plasma membrane, when expressedin Xenopus laevis oocytes. These proteins are thought to function in theregulation of the membrane potential and in transepithelial ionabsorption and secretion in the kidney. They possess two putativetransmembrane a-helical spanners (TMSs) with cytoplasmic N- andC-termini and a large luminal loop that may be glycosylated. The bovineprotein is 437 amino acyl residues in length and has the two putativeTMSs at positions 223-239 and 367-385. The human nuclear protein is muchsmaller (241 residues). A C. elegans homologue is 260 residues long.

[0056] Sugar Transporter

[0057] Organic substrates (sugars, amino acids, carboxylic acids andneutrotransmitters) are actively transported into eukaryotic cells byNa+ co-transport. Some of the transport proteins have beenidentified—for example, intestinal brush border Na+/glucose andNa⁺/proline transporters and the brain Na+/CI-/GABA transporter—andprogress has been made in locating their active sites and probing theirconformational states. The archetypical Na+-driven transporter is theintestinal brush border Na+/glucose co-transporter, and a defect in theco-transporter is the origin of the congenital glucose-galactosemalabsorption syndrome.

[0058] Cotransporters are a major class of membrane proteins—typicallywith 13 menbrane spanning helices. They cause the concentration ofmolecules across a membrane—nutrients, neurotransmitters, osmolytes andions. For example there are co transporters for amino acids, sugars,nucleosides and vitamins.

[0059] Na+/glucose co transporter (SGLT1 ) was reported in 1960 by BobCrane. Sodium dependent glucose transport occurs in both the kidney andthe intestine of animals. Both of these transporters show a closesimilarity to each other.

[0060] These transporters are reported to be multifunctional and havebeen shown to operate in 4 ways: 1) Uncoupled passive Na+ transport, 2)Downhill water transport, 3) Na+ and substrate transport, 4) Na+, waterand substrate transport. For further information regarding to thepresent invention, see Matsuo et al., Biochem Biophys Res Commun Sep. 8,1997 ;238(1):126-9.

[0061] Hexose transport into mammalian cells is catalyzed by members ofa small family of 44- to 55-kD membrane proteins that have specificfunctions and differ in their tissue distribution. Observed hexosetransporters have 12 membrane-spanning helices and a number of criticalconserved residues. By EST database searching for clones containingconserved GLUT sequences, followed by screening of rat tissues and5-prime RACE, Ibberson et al. and Doege et al. identified rodent andhuman cDNAs encoding a novel glucose transporter. (Ibberson, M., et al.,J. Biol. Chem. 275: 4607-4612, 2000, PubMed ID: 10671487; and Doege, H.,et al., J. Biol. Chem. 275: 16275-16280, 2000, PubMed ID: 10821868) Thehuman cDNA encodes a deduced 477-amino acid protein, designated GLUT8 orGLUTX1, that shares 85% sequence homology with the mouse sequence.Ibberson et al. found that the approximately 37-kD rat Glutx1 expressedin frog oocytes is unable to take up glucose unless the N-terminaldileucine motif, which may serve as an internalization signal, ismutated to alanines. Immunofluorescence analysis demonstrated thatGlutx1 is expressed intracellularly, whereas Glutx1(LL-AA) is expressedon the plasma membrane. In apparent contrast, Doege et al. found thatmembrane preparations from cells expressing GLUT8 cannot bindcytochalasin B in the presence of glucose and, when reconstituted inliposomes, have increased D-glucose transport activity. By Western blotanalysis, Doege et al. determined that human GLUT8 is expressed as a42-kD protein. Northern blot analysis revealed expression of a 2.4-kbtranscript, with strongest expression in testis and moderate expressionin other tissues except thyroid. In addition, Doege et al. found thatGLUT8 was not detectable in 2 patients with testicular carcinoma or intesticular tissue of 4 patients treated with estrogen. They found thatGlut8 mRNA was detectable in testis from pubertal and adult, but notprepubertal, rats

[0062] Glucose transport activity in early preimplantation mouse embryoshad been attributed to the known facilitative glucose transporters GLUT1(SLC2A1; 138140), GLUT2 (SLC2A2; 138160), and GLUT3 (SLC2A3; 138170).GLUT1 is present throughout the preimplantation period, which beginswith the 1-cell embryo and ends with the blastocyst stage. GLUT2 andGLUT3 are first expressed at a late 8-cell stage and remain present forthe rest of the preimplantation period. The simultaneous appearance ofall 3 transporters corresponds to the critical time in mammaliandevelopment when an embryonic fuel metabolism switches from theoxidation of lactate and pyruvate via the Krebs cycle and oxidativephosphorylation to anaerobic metabolism of glucose via glycolysis.Mammalian preimplantation blastocysts exhibit insulin-stimulated glucoseuptake despite the absence of the only known insulin-regulatedtransporter, GLUT4 (SLC2A4; 138190). Carayannopoulos et al. found thatmouse Glut8 exhibits 20 to 25% amino acid sequence identity with Glutl,Glut3, and Glut4. (Carayannopoulos, M. O., et al., Proc. Nat. Acad. Sci.97: 7313-7318, 2000, PubMed ID: 10860996) Insulin induced a change inthe intracellular localization of this protein, which translated intoincreased glucose uptake into the blastocyst, a process that wasinhibited by antisense oligoprobes. The presence of this transporter maybe necessary for successful blastocyst development, fuel metabolism, andsubsequent implantation. The existence of an alternative transporter mayexplain examples in other tissues of insulin-regulated glucose transportin the absence of Glut4

[0063] Doege et al. noted that the International Radiation HybridMapping Consortium localized the GLUT8 gene to chromosome 9 (A005N15).

[0064] Transporter proteins, particularly members of the sugartransporter subfamily, are a major target for drug action anddevelopment. Accordingly, it is valuable to the field of pharmaceuticaldevelopment to identify and characterize previously unknown transportproteins. The present invention advances the state of the art byproviding previously unidentified human transport proteins.

SUMMARY OF THE INVENTION

[0065] The present invention is based in part on the identification ofamino acid sequences of human transporter peptides and proteins that arerelated to the sugar transporter subfamily, as well as allelic variantsand other mammalian orthologs thereof. These unique peptide sequences,and nucleic acid sequences that encode these peptides, can be used asmodels for the development of human therapeutic targets, aid in theidentification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate transporteractivity in cells and tissues that express the transporter. Experimentaldata as provided in FIG. 1 indicates expression in ovary (adenocarcinomatissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancertissue (hypemephroma), germinal center B cell, colon, and infant brain.

DESCRIPTION OF THE FIGURE SHEETS

[0066]FIG. 1 provides the nucleotide sequence of cDNA molecules ortranscript sequences that encode the transporter proteins of the presentinvention. In addition structure and functional information is provided,such as ATG start, stop and tissue distribution, where available, thatallows one to readily determine specific uses of inventions based onthis molecular sequence. Experimental data as provided in FIG. 1indicates expression in ovary (adenocarcinoma tissue), uterus(leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue(hypemephroma), germinal center B cell, colon, and infant brain.

[0067]FIG. 2 provides the predicted amino acid sequence of thetransporter of the present invention. In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

[0068]FIG. 3 provides genomic sequences that span the gene encoding thetransporter protein of the present invention. In addition structure andfunctional information, such as intron/exon structure, promoterlocation, etc., is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.As illustrated in FIG. 3, SNPs, including insertion/deletion variants(“indels”), were identified at 42 different nucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

[0069] General Description

[0070] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a transporter protein or part of atransporter protein and are related to the sugar transporter subfamily.Utilizing these sequences, additional genomic sequences were assembledand transcript and/or cDNA sequences were isolated and characterized.Based on this analysis, the present invention provides amino acidsequences of human transporter peptides and proteins that are related tothe sugar transporter subfamily, nucleic acid sequences in the form oftranscript sequences, cDNA sequences and/or genomic sequences thatencode these transporter peptides and proteins, nucleic acid variation(allelic information), tissue distribution of expression, andinformation about the closest art known protein/peptide/domain that hasstructural or sequence homology to the transporter of the presentinvention.

[0071] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known transporter proteins ofthe sugar transporter subfamily and the expression pattern observed.Experimental data as provided in FIG. 1 indicates expression in ovary(adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney,kidney cancer tissue (hypemephroma), germinal center B cell, colon, andinfant brain. The art has clearly established the commercial importanceof members of this family of proteins and proteins that have expressionpatterns similar to that of the present gene. Some of the more specificfeatures of the peptides of the present invention, and the uses thereof,are described herein, particularly in the Background of the Inventionand in the annotation provided in the Figures, and/or are known withinthe art for each of the known sugar transporter family or subfamily oftransporter proteins.

[0072] Specific Embodiments

[0073] Peptide Molecules

[0074] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thetransporter family of proteins and are related to the sugar transportersubfamily (protein sequences are provided in FIG. 2, transcript/cDNAsequences are provided in FIG. 1 and genomic sequences are provided inFIG. 3). The peptide sequences provided in FIG. 2, as well as theobvious variants described herein, particularly allelic variants asidentified herein and using the information in FIG. 3, will be referredherein as the transporter peptides of the present invention, transporterpeptides, or peptides/proteins of the present invention.

[0075] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprising theamino acid sequences of the transporter peptides disclosed in the FIG.2, (encoded by the nucleic acid molecule shown in FIG. 1,transcript/cDNA or FIG. 3, genomic sequence), as well as all obviousvariants of these peptides that are within the art to make and use. Someof these variants are described in detail below.

[0076] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0077] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0078] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thetransporter peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0079] The isolated transporter peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix,kidney, kidney cancer tissue (hypernephroma), germinal center B cell,colon, and infant brain. For example, a nucleic acid molecule encodingthe transporter peptide is cloned into an expression vector, theexpression vector introduced into a host cell and the protein expressedin the host cell. The protein can then be isolated from the cells by anappropriate purification scheme using standard protein purificationtechniques. Many of these techniques are described in detail below.

[0080] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO:3 and SEQ IDNO:4), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO:1 and SEQ ID NO:2) and the genomicsequences provided in FIG. 3 (SEQ ID NO:5). The amino acid sequence ofsuch a protein is provided in FIG. 2. A protein consists of an aminoacid sequence when the amino acid sequence is the final amino acidsequence of the protein.

[0081] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:3and SEQ ID NO:4), for example, proteins encoded by the transcript/cDNAnucleic acid sequences shown in FIG. 1 (SEQ ID NO:1 and SEQ ID NO:2) andthe genomic sequences provided in FIG. 3 (SEQ ID NO:5). A proteinconsists essentially of an amino acid sequence when such an amino acidsequence is present with only a few additional amino acid residues, forexample from about 1 to about 100 or so additional residues, typicallyfrom 1 to about 20 additional residues in the final protein.

[0082] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO:3 and SEQ ID NO:4),for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO:1 and SEQ ID NO:2) and the genomicsequences provided in FIG. 3 (SEQ ID NO:5). A protein comprises an aminoacid sequence when the amino acid sequence is at least part of the finalamino acid sequence of the protein. In such a fashion, the protein canbe only the peptide or have additional amino acid molecules, such asamino acid residues (contiguous encoded sequence) that are naturallyassociated with it or heterologous amino acid residues/peptidesequences. Such a protein can have a few additional amino acid residuesor can comprise several hundred or more additional amino acids. Thepreferred classes of proteins that are comprised of the transporterpeptides of the present invention are the naturally occurring matureproteins. A brief description of how various types of these proteins canbe made/isolated is provided below.

[0083] The transporter peptides of the present invention can be attachedto heterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a transporter peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the transporter peptide. “Operativelylinked” indicates that the transporter peptide and the heterologousprotein are fused in-frame. The heterologous protein can be fused to theN-terminus or C-terminus of the transporter peptide.

[0084] In some uses, the fusion protein does not affect the activity ofthe transporter peptide per se. For example, the fusion protein caninclude, but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant transporter peptide. In certain host cells (e.g., mammalianhost cells), expression and/or secretion of a protein can be increasedby using a heterologous signal sequence.

[0085] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A transporter peptide-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the transporter peptide.

[0086] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0087] Such variantsican readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the transporter peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0088] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0089] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined:usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been. incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

[0090] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0091] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the transporter peptides of the present invention as well asbeing encoded by the same genetic locus as the transporter peptideprovided herein. As indicated by the data presented in FIG. 3, the mapposition was determined to be on chromosome 1.

[0092] Allelic variants of a transporter peptide can readily beidentified as being a human protein having a high degree (significant)of sequence homology/identity to at least a portion of the transporterpeptide as well as being encoded by the same genetic locus as thetransporter peptide provided herein. Genetic locus can readily bedetermined based on the genomic information provided in FIG. 3, such asthe genomic sequence mapped to the reference human. As indicated by thedata presented in FIG. 3, the map position was determined to be onchromosome 1. As used herein, two proteins (or a region of the proteins)have significant homology when the amino acid sequences are typically atleast about 70-80%, 80-90%, and more typically at least about 90-95% ormore homologous. A significantly homologous amino acid sequence,according to the present invention, will be encoded by a nucleic acidsequence that will hybridize to a transporter peptide encoding nucleicacid molecule under stringent conditions as more fully described below.

[0093]FIG. 3 provides information on SNPs that have been found in thegene encoding the transporter protein of the present invention. SNPswere identified at 42 different nucleotide positions in introns andregions 5′ and 3′ of the ORF. Such SNPs in introns and outside the ORFmay affect control/regulatory elements. Two SNPs in exons, of which 1 ofthese cause changes in the amino acid sequence (i.e., nonsynbnymousSNPs). The changes in the amino acid sequence that these SNPs cause isindicated in FIG. 3 and can readily be determined using the universalgenetic code and the protein sequence provided in FIG. 2 as a reference.

[0094] Paralogs of a transporter peptide can readily be identified ashaving some degree of significant sequence homology/identity to at leasta portion of the transporter peptide, as being encoded by a gene fromhumans, and as having similar activity or function. Two proteins willtypically be considered paralogs when the amino acid sequences aretypically at least about 60% or greater, and more typically at leastabout 70% or greater homology through a given region or domain. Suchparalogs will be encoded by a nucleic acid sequence that will hybridizeto a transporter peptide encoding nucleic acid molecule under moderateto stringent conditions as more fully described below.

[0095] Orthologs of a transporter peptide can readily be identified ashaving some degree of significant sequence homology/identity to at leasta portion of the transporter peptide as well as being encoded by a genefrom another organism. Preferred orthologs will be isolated frommammals, preferably primates, for the development of human therapeutictargets and agents. Such orthologs will be encoded by a nucleic acidsequence that will hybridize to a transporter peptide encoding nucleicacid molecule under moderate to stringent conditions, as more fullydescribed below, depending on the degree of relatedness of the twoorganisms yielding the proteins.

[0096] Non-naturally occurring variants of the transporter peptides ofthe present invention can readily be generated using recombinanttechniques. Such variants include, but are not limited to deletions,additions and substitutions in the amino acid sequence of thetransporter peptide. For example, one class of substitutions areconserved amino acid substitution. Such substitutions are those thatsubstitute agiven amino acid in a transporter peptide by another aminoacid of like characteristics. Typically seen as conservativesubstitutions are the replacements, one for another, among the aliphaticamino acids Ala, Val, leu, and Ile; interchange of the hydroxyl residuesSerand Thr, exchange of the acidic residues Asp and Glu; substitutionbetween the amide residues Asn and Gln; exchange of the basic residuesLys and Arg; and replacements among the aromatic residues Phe and Tyr.Guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

[0097] Variant transporter peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind ligand, abilityto transport ligand, ability to mediate signaling, etc. Fully functionalvariants typically contain only conservative variation or variation innon-critical residues or in non-critical regions. FIG. 2 provides theresult of protein analysis and can be used to identify criticaldomains/regions. Functional variants can also contain substitution ofsimilar amino acids that result in no change or an insignificant changein function. Alternatively, such substitutions may positively ornegatively affect function to some degree.

[0098] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0099] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as transporter activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0100] The present invention further provides fragments of thetransporter peptides, in addition to proteins and peptides that compriseand consist of such fragments, particularly those comprising theresidues identified in FIG. 2. The fragments to which the inventionpertains, however, are not to be construed as encompassing fragmentsthat may be disclosed publicly prior to the present invention.

[0101] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a transporter peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the transporter peptide or could be chosenfor the ability to perform a function, e.g. bind a substrate or act asan immunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe transporter peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0102] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally intransporter peptides are described in basic texts, detailed monographs,and the research literature, and they are well known to those of skillin the art (some of these features are identified in FIG. 2).

[0103] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0104] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth, Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y Acad. Sci. 663:48-62(1992)).

[0105] Accordingly, the transporter peptides of the present inventionalso encompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature transporter peptide is fused withanother compound, such as a compound to increase the half-life of thetransporter peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature transporter peptide, suchas a leader or secretory sequence or a sequence for purification of themature transporter peptide or a pro-protein sequence.

[0106] Protein/Peptide Uses

[0107] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in atransporter-effector protein interaction or transporter-ligandinteraction), the protein can be used to identify the bindingpartner/ligand so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these uses are capable of beingdeveloped into reagent grade or kit format for commercialization ascommercial products.

[0108] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0109] Substantial chemical and structural homology exists between thesugar transporter protein described herein and sugar transporterexpressed in the neonatal mouse hippocampus (see FIG. 1). As discussedin the background, sugar transporter expressed in the neonatal mousehippocampus are known in the art to be involved in sugar absorption.Accordingly, the sugar transporter protein and the encoding gene,provided by the present invention is useful for treating, preventing,and/or diagnosing neuropsychatric disorders, sigar malabsorption andother disorders associated with hippocampus sugar transporter.

[0110] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, transporters isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the transporter. Experimental data asprovided in FIG. 1 indicates that the transporter protein of the presentinvention is expressed in the ovary (adenocarcinoma tissue), uterus(leiomyosarcoma tissue), cervix, kidney cancer tissue (hypernephroma),germinal center B cell, colon, and infant brain by a virtual northernblot. In addition, PCR-based tissue screening panels indicate expressionin kidney. A large percentage of pharmaceutical agents are beingdeveloped that modulate the activity of transporter proteins,particularly members of the sugar transporter subfamily (see Backgroundof the Invention). The structural and functional information provided inthe Background and Figures provide specific and substantial uses for themolecules of the present invention, particularly in combination with theexpression information provided in FIG. 1. Experimental data as providedin FIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus(leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue(hypernephroma), germinal center B cell, colon, and infant brain. Suchuses can readily be determined using the information provided herein,that known in the art and routine experimentation.

[0111] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to transporters that arerelated to members of the sugar transporter subfamily. Such assaysinvolve any of the known transporter functions or activities orproperties useful for diagnosis and treatment of transporter-relatedconditions that are specific for the subfamily of transporters that theone of the present invention belongs to, particularly in cells andtissues that express the transporter. Experimental data as provided inFIG. 1 indicates that the transporter protein of the present inventionis expressed in the ovary (adenocarcinoma tissue), uterus(leiomyosarcoma tissue), cervix, kidney cancer tissue (hypernephroma),germinal center B cell, colon, and infant brain by a virtual northernblot. In addition, PCR-based tissue screening panels indicate expressionin kidney. The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems ((Hodgson,Bio/technology, Sep. 10, 1992, (9);973-80). Cell-based systems can benative, i.e., cells that normally express the transporter, as a biopsyor expanded in cell culture. Experimental data as provided in FIG. 1indicates expression in ovary (adenocarcinoma tissue), uterus(leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue(hypernephroma), germinal center B cell, colon, and infant brain. In analternate embodiment, cell-based assays involve recombinant host cellsexpressing the transporter protein.

[0112] The polypeptides can be used to identify compounds that modulatetransporter activity of the protein in its natural state or an alteredform that causes a specific disease or pathology associated with thetransporter. Both the transporters of the present invention andappropriate variants and fragments can be used in high-throughputscreens to assay candidate compounds for the ability to bind to thetransporter. These compounds can be further screened against afunctional transporter to determine the effect of the compound on thetransporter activity. Further, these compounds can be tested in animalor invertebrate systems to determine activity/effectiveness. Compoundscan be identified that activate (agonist) or inactivate (antagonist) thetransporter to a desired degree.

[0113] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the transporter protein and a molecule that normally interactswith the transporter protein, e.g. a substrate or a component of thesignal pathway that the transporter protein normally interacts (forexample, another transporter). Such assays typically include the stepsof combining the transporter protein with a candidate compound underconditions that allow the transporter protein, or fragment, to interactwith the target molecule, and to detect the formation of a complexbetween the protein and the target or to detect the biochemicalconsequence of the interaction with the transporter protein and thetarget, such as any of the associated effects of signal transductionsuch as changes in membrane potential, protein phosphorylation, cAMPturnover, and adenylate cyclase activation, etc.

[0114] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L- configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0115] One candidate compound is a soluble fragment of the receptor thatcompetes for ligand binding. Other candidate compounds include mutanttransporters or appropriate fragments containing mutations that affecttransporter function and thus compete for ligand. Accordingly, afragment that competes for ligand, for example with a higher affinity,or a fragment that binds ligand but does not allow release, isencompassed by the invention.

[0116] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) transporter activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate transporter activity. Thus, the transport of aligand, change in cell membrane potential, activation of a protein, achange in the expression of genes that are up- or down-regulated inresponse to the transporter protein dependent signal cascade can beassayed.

[0117] Any of the biological or biochemical functions mediated by thetransporter can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the transporter can be assayed.Experimental data as provided in FIG. 1 indicates that the transporterprotein of the present invention is expressed in the ovary(adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidneycancer tissue (hypemephroma), germinal center B cell, colon, and infantbrain by a virtual northern blot. In addition, PCR-based tissuescreening panels indicate expression in kidney.

[0118] Binding andlor activating compounds can also be screened by usingchimeric transporter proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a ligand-binding region can be usedthat interacts with a different ligand then that which is recognized bythe native transporter. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the transporter is derived.

[0119] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the transporter (e.g. binding partners and/orligands). Thus, a compound is exposed to a transporter polypeptide underconditions that allow the compound to bind or to otherwise interact withthe polypeptide. Soluble transporter polypeptide is also added to themixture. If the test compound interacts with the soluble transporterpolypeptide, it decreases the amount of complex formed or activity fromthe transporter target. This type of assay is particularly useful incases in which compounds are sought that interact with specific regionsof the transporter. Thus, the soluble polypeptide that competes with thetarget transporter region is designed to contain peptide sequencescorresponding to the region of interest.

[0120] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the transporter protein, or fragment, orits target molecule to facilitate separation of complexes fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay.

[0121] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of transporter-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a transporter-binding protein and a candidate compound are incubatedin the transporter protein-presenting wells and the amount of complextrapped in the well can be quantitated. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the transporter protein target molecule, or which arereactive with transporter protein and compete with the target molecule,as well as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the target molecule.

[0122] Agents that modulate one of the transporters of the presentinvention can be identified using one or more of the above assays, aloneor in combination. It is generally preferable to use a cell-based orcell free system first and then confirm activity in an animal or othermodel system. Such model systems are well known in the art and canreadily be employed in this context.

[0123] Modulators of transporter protein activity identified accordingto these drug screening assays can be used to treat a subject with adisorder mediated by the transporter pathway, by treating cells ortissues that express the transporter. Experimental data as provided inFIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus(leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue(hypernephroma), germinal center B cell, colon, and infant brain. Thesemethods of treatment include the steps of administering a modulator oftransporter activity in a pharmaceutical composition to a subject inneed of such treatment, the modulator being identified as describedherein.

[0124] In yet another aspect of the invention, the transporter proteinscan be used as “bait proteins” in a two-hybrid assay or three-hybridassay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the transporter and are involved in transporteractivity. Such transporter-binding proteins are also likely to beinvolved in the propagation of signals by the transporter proteins ortransporter targets as, for example, downstream elements of atransporter-mediated signaling pathway. Alternatively, suchtransporter-binding proteins are likely to be transporter inhibitors.

[0125] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a transporterprotein is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming atransporter-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. Thisproximity allows transcription of a reporter gene (e.g., LacZ) which isoperably linked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the transporter protein.

[0126] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a transporter-modulating agent, an antisensetransporter nucleic acid molecule, a transporter-specific antibody, or atransporter-binding partner) can be used in an animal or other model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal or other model to determine the mechanism of action ofsuch an agent. Furthermore, this invention pertains to uses of novelagents identified by the above-described screening assays for treatmentsas described herein.

[0127] The transporter proteins of the present invention are also usefulto provide a target for diagnosing a disease or predisposition todisease mediated by the peptide. Accordingly, the invention providesmethods for detecting the presence, or levels of, the protein (orencoding mRNA) in a cell, tissue, or organism. Experimental data asprovided in FIG. 1 indicates expression in ovary (adenocarcinomatissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancertissue (hypemephroma), germinal center B cell, colon, and infant brain.The method involves contacting a biological sample with a compoundcapable of interacting with the transporter protein such that theinteraction can be detected. Such an assay can be provided in a singledetection format or a multi-detection format such as an antibody chiparray.

[0128] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0129] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,alteredtryptic peptide digest, altered transporter activity incell-based or cell-free assay, alteration in ligand or antibody-bindingpattern, altered isoelectric point, direct amino acid sequencing, andany other of the known assay techniques useful for detecting mutationsin a protein. Such an assay can be provided in a single detection formator a multi-detection format such as an antibody chip array.

[0130] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (EISAs), Western blots, immunoprecipitationsand immunofluorescence using a detection reagent, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody or other types of detection agent. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques. Particularlyuseful are methods that detect the allelic variant of a peptideexpressed in a subject and methods which detect fragments of a peptidein a sample.

[0131] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the transporter protein in which oneor more of the transporter functions in one population is different fromthose in another population. The peptides thus allow a target toascertain a genetic predisposition that can affect treatment modality.Thus, in a ligand-based treatment, polymorphism may give rise to aminoterminal extracellular domains and/or other ligand-binding regions thatare more or less active in ligand binding, and transporter activation.Accordingly, ligand dosage would necessarily be modified to maximize thetherapeutic effect within a given population containing a polymorphism.As an alternative to genotyping, specific polymorphic peptides could beidentified.

[0132] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in ovary (adenocarcinoma tissue), uterus (leiomyosarcomatissue), cervix, kidney, kidney cancer tissue (hypemephroma), germinalcenter B cell, colon, and infant brain. Accordingly, methods fortreatment include the use of the transporter protein or fragments.

[0133] Antibodies

[0134] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0135] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0136] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0137] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0138] Antibodies are preferably prepared from regions or discretefragments of the transporter proteins. Antibodies can be prepared fromany region of the peptide as described herein. However, preferredregions will include those involved in function/activity and/ortransporter/binding partner interaction. FIG. 2 can be used to identifyparticularly important regions while sequence alignment can be used toidentify conserved and unique sequence fragments.

[0139] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0140] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0141] Antibody Uses

[0142] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat the transporter protein of the present invention is expressed inthe ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue),cervix, kidney cancer tissue (hypernephroma), germinal center B cell,colon, and infant brain by a virtual northern blot. In addition,PCR-based tissue screening panels indicate expression in kidney.Further, such antibodies can be used to detect protein in situ, invitro, or in a cell lysate or supernatant in order to evaluate theabundance and pattern of expression. Also, such antibodies can be usedto assess abnormal tissue distribution or abnormal expression duringdevelopment or progression of a biological condition. Antibody detectionof circulating fragments of the full length protein can be used toidentify turnover.

[0143] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in ovary (adenocarcinoma tissue), uterus(leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue(hypernephroma), germinal center B cell, colon, and infant brain. If adisorder is characterized by a specific mutation in the protein,antibodies specific for this mutant protein can be used to assay for thepresence of the specific mutant protein.

[0144] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in ovary(adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney,kidney cancer tissue (hypernephroma), germinal center B cell, colon, andinfant brain. The diagnostic uses can be applied, not only in genetictesting, but also in monitoring a treatment modality. Accordingly, wheretreatment is ultimately aimed at correcting expression level or thepresence of aberrant sequence and aberrant tissue distribution ordevelopmental expression, antibodies directed against the protein orrelevant fragments can be used to monitor therapeutic efficacy.

[0145] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0146] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in ovary (adenocarcinomatissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancertissue (hypemephroma), germinal center B cell, colon, and infant brain.Thus, where a specific protein has been correlated with expression in aspecific tissue, antibodies that are specific for this protein can beused to identify a tissue type.

[0147] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the transporter peptide to abinding partner such as a ligand or protein binding partner. These usescan also be applied in a therapeutic context in which treatment involvesinhibiting the protein's function. An antibody can be used, for example,to block binding, thus modulating (agonizing or antagonizing) thepeptides activity. Antibodies can be prepared against specific fragmentscontaining sites required for function or against intact protein that isassociated with a cell or cell membrane. See FIG. 2 for structuralinformation relating to the proteins of the present invention.

[0148] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nucleic acid arrays and similar methods have been developedfor antibody arrays.

[0149] Nucleic Acid Molecules

[0150] The present invention further provides isolated nucleic acidmolecules that encode a transporter peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the transporter peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0151] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences that naturally flank the nucleic acid (i.e., sequences locatedat the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of theorganism from which the nucleic acid is derived. However, there can besome flanking nucleotide sequences, for example up to about 5 KB, 4 KB,3 KB, 2 KB, or 1 KB or less, particularly contiguous peptide encodingsequences and peptide encoding sequences within the same gene butseparated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0152] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0153] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0154] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIGS. 1 or 3(SEQ ID NO:1 and SEQ ID NO:2, transcript sequences and SEQ ID NO:5,genomic sequence), or any nucleic acid molecule that encodes theproteins provided in FIG. 2, SEQ ID NO:3 and SEQ ID NO:4. A nucleic acidmolecule consists of a nucleotide sequence when the nucleotide sequenceis the complete nucleotide sequence of the nucleic acid molecule.

[0155] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIGS. 1 or3 (SEQ ID NO:1 and SEQ ID NO:2, transcript sequences and SEQ ID NO:5,genomic sequence), or any nucleic acid molecule that encodes theproteins provided in FIG. 2, SEQ ID NO:3 and SEQ ID NO:4. A nucleic acidmolecule consists essentially of a nucleotide sequence when such anucleotide sequence is present with only a few additional nucleic acidresidues in the final nucleic acid molecule.

[0156] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIGS. 1 or 3 (SEQ IDNO:1 and SEQ ID NO:2, transcript sequences and SEQ ID NO:5, genomicsequence), or any nucleic acid molecule that encodes the proteinsprovided in FIG. 2, SEQ ID NO:3 and SEQ ID NO:4. A nucleic acid moleculecomprises a nucleotide sequence when the nucleotide sequence is at leastpart of the final nucleotide sequence of the nucleic acid molecule. Insuch a fashion, the nucleic acid molecule can be only the nucleotidesequence or have additional nucleic acid residues, such as nucleic acidresidues that are naturally associated with it or heterologousnucleotide sequences. Such a nucleic acid molecule can have a fewadditional nucleotides or can comprise several hundred or moreadditional nucleotides. A brief description of how various types ofthese nucleic acid molecules can be readily made/isolated is providedbelow.

[0157] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0158] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0159] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the transporter peptidealone, the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0160] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0161] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the transporter proteinsof the present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0162] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0163] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0164] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide. pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0165] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. As indicated by thedata presented in FIG. 3, the map position was determined to be onchromosome 1.

[0166]FIG. 3 provides information on SNPs that have been found in thegene encoding the transporter protein of the present invention. SNPswere identified at 42 different nucleotide positions in introns andregions 5′ and 3′ of the ORF. Such SNPs in introns and outside the ORFmay affect control/regulatory elements. Two SNPs in exons, of which 1 ofthese cause changes in the amino acid sequence (i.e., nonsynonymousSNPs). The changes in the amino acid sequence that these SNPs cause isindicated in FIG. 3 and can readily be determined using the universalgenetic code and the protein sequence provided in FIG. 2 as a reference.

[0167] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45 C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

[0168] Nucleic Acid Molecule Uses

[0169] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA. and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in Figure,2. As illustrated in FIG. 3, SNPs, including insertion/deletion variants(“indels”), were identified at 42 different nucleotide positions.

[0170] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0171] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0172] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0173] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0174] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods. As indicated by the datapresented in FIG. 3, the map position was determined to be on chromosome1.

[0175] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0176] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0177] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0178] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0179] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0180] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that the transporter protein of the present invention isexpressed in the ovary (adenocarcinoma tissue), uterus (leiomyosarcomatissue), cervix, kidney cancer tissue (hypemephroma), germinal center Bcell, colon, and infant brain by a virtual northern blot.

[0181] Accordingly, the probes can be used to detect the presence of, orto determine levels of, a specific nucleic acid molecule in cells,tissues, and in organisms. The nucleic acid whose level is determinedcan be DNA or RNA. Accordingly, probes corresponding to the peptidesdescribed herein can be used to assess expression andlor gene copynumber in a given cell, tissue, or organism. These uses are relevant fordiagnosis of disorders involving an increase or decrease in transporterprotein expression relative to normal results.

[0182] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

[0183] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a transporter protein, such asby measuring a level of a transporter-encoding nucleic acid in a sampleof cells from a subject e.g., mRNA or genomic DNA, or determining if atransporter gene has been mutated. Experimental data as provided in FIG.1 indicates that the transporter protein of the present invention isexpressed in the ovary (adenocarcinoma tissue), uterus (leiomyosarcomatissue), cervix, kidney cancer tissue (hypemephroma), germinal center Bcell, colon, and infant brain by a virtual northern blot. In addition,PCR-based tissue screening panels indicate expression in kidney.

[0184] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate transporter nucleic acid expression.

[0185] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the transporter gene, particularly biological andpathological processes that are mediated by the transporter in cells andtissues that express it. Experimental data as provided in FIG. 1indicates expression in ovary (adenocarcinoma tissue), uterus(leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue(hypemephroma), germinal center B cell, colon, and infant brain. Themethod typically includes assaying the ability of the compound tomodulate the expression of the transporter nucleic acid and thusidentifying a compound that can be used to treat a disordercharacterized by undesired transporter nucleic acid expression. Theassays can be performed in cell-based and cell-free systems. Cell-basedassays include cells naturally expressing the transporter nucleic acidor recombinant cells genetically engineered to express specific nucleicacid sequences.

[0186] The assay for transporter nucleic acid expression can involvedirect assay of nucleic acid levels, such as mRNA levels, or oncollateral compounds involved in the signal pathway. Further, theexpression of genes that are up- or down-regulated in response to thetransporter protein signal pathway can also be assayed. In thisembodiment the regulatory regions of these genes can be operably linkedto a reporter gene such as luciferase.

[0187] Thus, modulators of transporter gene expression can be identifiedin a method wherein a cell is contacted with a candidate compound andthe expression of mRNA determined. The level of expression oftransporter mRNA in the presence of the candidate compound is comparedto the level of expression of transporter mRNA in the absence of thecandidate compound. The candidate compound can then be identified as amodulator of nucleic acid expression based on this comparison and beused, for example to treat a disorder characterized by aberrant nucleicacid expression. When expression of mRNA is statistically significantlygreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of nucleic acidexpression. When nucleic acid expression is statistically significantlyless in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of nucleic acidexpression.

[0188] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate transporter nucleic acidexpression in cells and tissues that express the transporter.Experimental data as provided in FIG. 1 indicates that the transporterprotein of the present invention is expressed in the ovary(adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidneycancer tissue (hypemephroma), germinal center B cell, colon, and infantbrain by a virtual northern blot. In addition, PCR-based tissuescreening panels indicate expression in kidney. Modulation includes bothup-regulation (i.e. activation or agonization) or down-regulation(suppression or antagonization) or nucleic acid expression.

[0189] Alternatively, a modulator for transporter nucleic acidexpression can be a small molecule or drug identified using thescreening assays described herein as long as the drug or small moleculeinhibits the transporter nucleic acid expression in the cells andtissues that express the protein. Experimental data as provided in FIG.1 indicates expression in ovary (adenocarcinoma tissue), uterus(leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue(hypernephroma), germinal center B cell, colon, and infant brain.

[0190] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe transporter gene in clinical trials or in a treatment regimen. Thus,the gene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

[0191] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in transporter nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in transporter genes andgene expression products such as mRNA. The nucleic acid molecules can beused as hybridization probes to detect naturally occurring geneticmutations in the transporter gene and thereby to determine whether asubject with the mutation is at risk for a disorder caused by themutation. Mutations include deletion, addition, or substitution of oneor more nucleotides in the gene, chromosomal rearrangement, such asinversion or transposition, modification of genomic DNA, such asaberrant methylation patterns or changes in gene copy number, such asamplification. Detection of a mutated form of the transporter geneassociated with a dysfunction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of a transporterprotein.

[0192] Individuals carrying mutations in the transporter gene can bedetected at the nucleic acid level by a variety of techniques. FIG. 3provides information on SNPs that have been found in the gene encodingthe transporter protein of the present invention. SNPs were identifiedat 42 different nucleotide positions in introns and regions 5′ and 3′ ofthe ORF. Such SNPs in introns and outside the ORF may affectcontrol/regulatory elements. Two SNPs in exons, of which 1 of thesecause changes in the amino acid sequence (i.e., nonsynonymous SNPs). Thechanges in the amino acid sequence that these SNPs cause is indicated inFIG. 3 and can readily be determined using the universal genetic codeand the protein sequence provided in FIG. 2 as a reference. As indicatedby the data presented in FIG. 3, the map position was determined to beon chromosome 1. Genomic DNA can be analyzed directly or can beamplified by using PCR prior to analysis. RNA or cDNA can be used in thesame way. In some uses, detection of the mutation involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat.Nos.4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS91:360-364 (1994)), the latter of which can be particularly useful fordetecting point mutations in the gene (see Abravaya et al., NucleicAcids Res. 23:675-682 (1995)). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

[0193] Alternatively, mutations in a transporter gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0194] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0195] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant transporter gene and a wild-type gene can be determined by directDNA sequencing. A variety of automated sequencing procedures can beutilized when performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0196] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 ;(1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl, 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gelelectrophoresis(Myers et al., Nature 313:495 (1985)). Examples of othertechniques for detecting point mutations include selectiveoligonucleotide hybridization, selective amplification, and selectiveprimer extension.

[0197] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the transporter gene in an individual in order to select anappropriate compound or dosage regimen for treatment. FIG. 3 providesinformation on SNPs that have been found in the gene encoding thetransporter protein of the present invention. SNPs were identified at 42different nucleotide positions in introns and regions 5′ and 3′ of theORF. Such SNPs in introns and outside the ORF may affectcontrol/regulatory elements. Two SNPs in exons, of which 1 of thesecause changes in the amino acid sequence (i.e., nonsynonymous SNPs). Thechanges in the amino acid sequence that these SNPs cause is indicated inFIG. 3 and can readily be determined using the universal genetic codeand the protein sequence provided in FIG. 2 as a reference.

[0198] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0199] The nucleic acid molecules are thus useful as antisenseconstructs to control transporter gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of transporter protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into transporter protein.

[0200] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of transporter nucleicacid. Accordingly, these molecules can treat a disorder characterized byabnormal or undesired transporter nucleic acid expression. Thistechnique involves cleavage by means of ribozymes containing nucleotidesequences complementary to one or more regions in the mRNA thatattenuate the ability of the mRNA to be translated. Possible regionsinclude coding regions and particularly coding regions corresponding tothe catalytic and other functional activities of the transporterprotein, such as ligand binding.

[0201] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in transporter geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desiredtransporter protein to treat the individual.

[0202] The invention also encompasses kits for detecting the presence ofa transporter nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that the transporter protein of the presentinvention is expressed in the ovary (adenocarcinoma tissue), uterus(leiomyosarcoma tissue), cervix, kidney cancer tissue (hypernephroma),germinal center B cell, colon, and infant brain by a virtual northernblot. In addition, PCR-based tissue screening panels indicate expressionin kidney. For example, the kit can comprise reagents such as a labeledor labelable nucleic acid or agent capable of detecting transporternucleic acid in a biological sample; means for determining the amount oftransporter nucleic acid in the sample; and means for comparing theamount of transporter nucleic acid in the sample with a standard. Thecompound or agent can be packaged in a suitable container. The kit canfurther comprise instructions for using the kit to detect transporterprotein mRNA or DNA.

[0203] Nucleic Acid Arrays

[0204] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ IDNOS:1, 2, and 5).

[0205] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0206] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides that cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0207] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0208] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot-blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0209] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0210] Using such arrays, the present invention provides methods toidentify the expression of the transporter proteins/peptides of thepresent invention. In detail, such methods comprise incubating a testsample with one or more nucleic acid molecules and assaying for bindingof the nucleic acid molecule with components within the test sample.Such assays will typically involve arrays comprising many genes, atleast one of which is a gene of the present invention and or alleles ofthe transporter gene of the present invention. FIG. 3 providesinformation on SNPs that have been found in the gene encoding thetransporter protein of the present invention. SNPs were identified at 42different nucleotide positions in introns and regions 5′ and 3′ of theORF. Such SNPs in introns and outside the ORF may affectcontrol/regulatory elements. Two SNPs in exons, of which 1 of thesecause changes in the amino acid sequence (i.e., nonsynonymous SNPs). Thechanges in the amino acid sequence that these SNPs cause is indicated inFIG. 3 and can readily be determined using the universal genetic codeand the protein sequence provided in FIG. 2 as a reference.

[0211] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridizationamplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1 982), Vol.2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0212] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0213] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0214] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0215] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified transporter gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

[0216] Vectors/Host Cells

[0217] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0218] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0219] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in procaryotic or eukaryoticcells or in both (shuttle vectors).

[0220] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0221] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0222] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0223] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0224] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0225] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0226] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0227] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0228] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors can.increase the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enterotransporter. Typical fusionexpression vectors include pGEX (Smith et al., Gene 67:3140 (1988)),pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein. Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology185:60-89 (1990)).

[0229] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0230] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0231] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., Mol. Cell. Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

[0232] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC Kaufman et al.,EMBO J. 6:187-195 (1987)).

[0233] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0234] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0235] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0236] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0237] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0238] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0239] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0240] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0241] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such astransporters, appropriate secretion signals are incorporated into thevector. The signal sequence can be endogenous to the peptides orheterologous to these peptides.

[0242] Where the peptide is not secreted into the medium, which istypically the case with transporters, the protein can be isolated fromthe host cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0243] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified-methionine in some cases as aresult of a host-mediated process.

[0244] Uses of Vectors and Host Cells

[0245] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga transporter protein or peptide that can be further purified to producedesired amounts of transporter protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0246] Host cells are also useful for conducting cell-based assaysinvolving the transporter protein or transporter protein fragments, suchas those described above as well as other formats known in the art.Thus, a recombinant host cell expressing a native transporter protein isuseful for assaying compounds that stimulate or inhibit transporterprotein function.

[0247] Host cells are also useful for identifying transporter proteinmutants in which these functions are affected. If the mutants naturallyoccur and give rise to a pathology, host cells containing the mutationsare useful to assay compounds that have a desired effect on the mutanttransporter protein (for example, stimulating or inhibiting function)which may not be indicated by their effect on the native transporterprotein.

[0248] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA that is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a transporterprotein and identifying and evaluating modulators of transporter proteinactivity. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, and amphibians.

[0249] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the transporter proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0250] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the transporter protein to particularcells.

[0251] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0252] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0253] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0254] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect ligand binding,transporter protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivotransporter protein function, including ligand interaction, the effectof specific mutant transporter proteins on transporter protein functionand ligand interaction, and the effect of chimeric transporter proteins.It is also possible to assess the effect of null mutations, that ismutations that substantially or completely eliminate one or moretransporter protein functions.

[0255] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 49 1 1473 DNA Homo sapiens 1 atgacccagg ggaagaagaa gaaacgggccgcgaaccgca gtatcatgct ggccaagaag 60 atcatcatta aggacggagg cacgcctcaaggaataggtt ctcctagtgt ctatcatgca 120 gttatcgtca tctttttgga gttttttgcttggggactat tgacagcacc caccttggtg 180 gtattacatg aaacctttcc taaacatacatttctgatga acggcttaat tcaaggagta 240 aagggtttgt tgtcattcct tagtgccccgcttattggtg ctctttctga tgtttggggc 300 cgaaaatcct tcttgctgct aacggtgtttttcacatgtg ccccaattcc tttaatgaag 360 atcagcccat ggtggtactt tgctgttatctctgtttctg gggtttttgc agtgactttt 420 tctgtggtat ttgcatacgt agcagatataacccaagagc atgaaagaag tatggcttat 480 ggactggttt cagcaacatt tgctgcaagtttagtcacca gtcctgcaat tggagcttat 540 cttggacgag tatatgggga cagcttggtggtggtcttag ctacagcaat agctttgcta 600 gatatttgtt ttatccttgt tgctgtgccagagtcgttgc ctgagaaaat gcggccagca 660 tcctggggag cacccatttc ctgggaacaagctgaccctt ttgcgtcctt aaaaaaagtc 720 ggccaagatt ccatagtgct gctgatctgcattacagtgt ttctctccta cctaccggag 780 gcaggccaat attccagctt ttttttatacctcagacaga taatgaaatt ttcaccagaa 840 agtgttgcag cgtttatagc agtccttggcattctttcca ttattgcaca gaccatagtc 900 ttgagtttac ttatgaggtc aattggaaataagaacacca ttttactggg tctaggattt 960 caaatattac agttggcatg gtatggctttggttcagaac cttggatgat gtgggctgct 1020 ggggcagtag cagccatgtc tagcatcacctttcctgctg tcagtgcact tgtttcacga 1080 actgctgatg ctgatcaaca gggtgtcgttcaaggaatga taacaggaat tcgaggatta 1140 tgcaatggtc tgggaccggc cctctatggattcattttct acatattcca tgtggaactt 1200 aaagaactgc caataacagg aacagacttgggaacaaaca caagccctca gcaccacttt 1260 gaacagaatt ccatcatccc tggccctcccttcctatttg gagcctgttc agtactgctg 1320 gctctgcttg ttgccttgtt tattccggaacataccaatt taagcttaag gtccagcagt 1380 tggagaaagc actgtggcag tcacagccatcctcataata cacaagcgcc aggagaggcc 1440 aaagaacctt tactccagga cacaaatgtgtga 1473 2 1377 DNA Homo sapiens 2 atgacccagg ggaagaagaa gaaacgggccgcgaaccgca gtatcatgct ggccaagaag 60 atcatcatta aggacggagg cacggtattacatgaaacct ttcctaaaca tacatttctg 120 atgaacggct taattcaagg agtaaagggtttgttgtcat tccttagtgc cccgcttatt 180 ggtgctcttt ctgatgtttg gggccgaaaatccttcttgc tgctaacggt gtttttcaca 240 tgtgccccaa ttcctttaat gaagatcagcccatggtggt actttgctgt tatctctgtt 300 tctggggttt ttgcagtgac tttttctgtggtatttgcat acgtagcaga tataacccaa 360 gagcatgaaa gaagtatggc ttatggactggtttcagcaa catttgctgc aagtttagtc 420 accagtcctg caattggagc ttatcttggacgagtatatg gggacagctt ggtggtggtc 480 ttagctacag caatagcttt gctagatatttgttttatcc ttgttgctgt gccagagtcg 540 ttgcctgaga aaatgcggcc agcatcctggggagcaccca tttcctggga acaagctgac 600 ccttttgcgt ccttaaaaaa agtcggccaagattccatag tgctgctgat ctgcattaca 660 gtgtttctct cctacctacc ggaggcaggccaatattcca gctttttttt atacctcaga 720 cagataatga aattttcacc agaaagtgttgcagcgttta tagcagtcct tggcattctt 780 tccattattg cacagaccat agtcttgagtttacttatga ggtcaattgg aaataagaac 840 accattttac tgggtctagg atttcaaatattacagttgg catggtatgg ctttggttca 900 gaaccttgga tgatgtgggc tgctggggcagtagcagcca tgtctagcat cacctttcct 960 gctgtcagtg cacttgtttc acgaactgctgatgctgatc aacagggtgt cgttcaagga 1020 atgataacag gaattcgagg attatgcaatggtctgggac cggccctcta tggattcatt 1080 ttctacatat tccatgtgga acttaaagaactgccaataa caggaacaga cttgggaaca 1140 aacacaagcc ctcagcacca ctttgaacagaattccatca tccctggccc tcccttccta 1200 tttggagcct gttcagtact gctggctctgcttgttgcct tgtttattcc ggaacatacc 1260 aatttaagct taaggtccag cagttggagaaagcactgtg gcagtcacag ccatcctcat 1320 aatacacaag cgccaggaga ggccaaagaacctttactcc aggacacaaa tgtgtga 1377 3 490 PRT Homo sapiens 3 Met Thr GlnGly Lys Lys Lys Lys Arg Ala Ala Asn Arg Ser Ile Met 1 5 10 15 Leu AlaLys Lys Ile Ile Ile Lys Asp Gly Gly Thr Pro Gln Gly Ile 20 25 30 Gly SerPro Ser Val Tyr His Ala Val Ile Val Ile Phe Leu Glu Phe 35 40 45 Phe AlaTrp Gly Leu Leu Thr Ala Pro Thr Leu Val Val Leu His Glu 50 55 60 Thr PhePro Lys His Thr Phe Leu Met Asn Gly Leu Ile Gln Gly Val 65 70 75 80 LysGly Leu Leu Ser Phe Leu Ser Ala Pro Leu Ile Gly Ala Leu Ser 85 90 95 AspVal Trp Gly Arg Lys Ser Phe Leu Leu Leu Thr Val Phe Phe Thr 100 105 110Cys Ala Pro Ile Pro Leu Met Lys Ile Ser Pro Trp Trp Tyr Phe Ala 115 120125 Val Ile Ser Val Ser Gly Val Phe Ala Val Thr Phe Ser Val Val Phe 130135 140 Ala Tyr Val Ala Asp Ile Thr Gln Glu His Glu Arg Ser Met Ala Tyr145 150 155 160 Gly Leu Val Ser Ala Thr Phe Ala Ala Ser Leu Val Thr SerPro Ala 165 170 175 Ile Gly Ala Tyr Leu Gly Arg Val Tyr Gly Asp Ser LeuVal Val Val 180 185 190 Leu Ala Thr Ala Ile Ala Leu Leu Asp Ile Cys PheIle Leu Val Ala 195 200 205 Val Pro Glu Ser Leu Pro Glu Lys Met Arg ProAla Ser Trp Gly Ala 210 215 220 Pro Ile Ser Trp Glu Gln Ala Asp Pro PheAla Ser Leu Lys Lys Val 225 230 235 240 Gly Gln Asp Ser Ile Val Leu LeuIle Cys Ile Thr Val Phe Leu Ser 245 250 255 Tyr Leu Pro Glu Ala Gly GlnTyr Ser Ser Phe Phe Leu Tyr Leu Arg 260 265 270 Gln Ile Met Lys Phe SerPro Glu Ser Val Ala Ala Phe Ile Ala Val 275 280 285 Leu Gly Ile Leu SerIle Ile Ala Gln Thr Ile Val Leu Ser Leu Leu 290 295 300 Met Arg Ser IleGly Asn Lys Asn Thr Ile Leu Leu Gly Leu Gly Phe 305 310 315 320 Gln IleLeu Gln Leu Ala Trp Tyr Gly Phe Gly Ser Glu Pro Trp Met 325 330 335 MetTrp Ala Ala Gly Ala Val Ala Ala Met Ser Ser Ile Thr Phe Pro 340 345 350Ala Val Ser Ala Leu Val Ser Arg Thr Ala Asp Ala Asp Gln Gln Gly 355 360365 Val Val Gln Gly Met Ile Thr Gly Ile Arg Gly Leu Cys Asn Gly Leu 370375 380 Gly Pro Ala Leu Tyr Gly Phe Ile Phe Tyr Ile Phe His Val Glu Leu385 390 395 400 Lys Glu Leu Pro Ile Thr Gly Thr Asp Leu Gly Thr Asn ThrSer Pro 405 410 415 Gln His His Phe Glu Gln Asn Ser Ile Ile Pro Gly ProPro Phe Leu 420 425 430 Phe Gly Ala Cys Ser Val Leu Leu Ala Leu Leu ValAla Leu Phe Ile 435 440 445 Pro Glu His Thr Asn Leu Ser Leu Arg Ser SerSer Trp Arg Lys His 450 455 460 Cys Gly Ser His Ser His Pro His Asn ThrGln Ala Pro Gly Glu Ala 465 470 475 480 Lys Glu Pro Leu Leu Gln Asp ThrAsn Val 485 490 4 458 PRT Homo sapiens 4 Met Thr Gln Gly Lys Lys Lys LysArg Ala Ala Asn Arg Ser Ile Met 1 5 10 15 Leu Ala Lys Lys Ile Ile IleLys Asp Gly Gly Thr Val Leu His Glu 20 25 30 Thr Phe Pro Lys His Thr PheLeu Met Asn Gly Leu Ile Gln Gly Val 35 40 45 Lys Gly Leu Leu Ser Phe LeuSer Ala Pro Leu Ile Gly Ala Leu Ser 50 55 60 Asp Val Trp Gly Arg Lys SerPhe Leu Leu Leu Thr Val Phe Phe Thr 65 70 75 80 Cys Ala Pro Ile Pro LeuMet Lys Ile Ser Pro Trp Trp Tyr Phe Ala 85 90 95 Val Ile Ser Val Ser GlyVal Phe Ala Val Thr Phe Ser Val Val Phe 100 105 110 Ala Tyr Val Ala AspIle Thr Gln Glu His Glu Arg Ser Met Ala Tyr 115 120 125 Gly Leu Val SerAla Thr Phe Ala Ala Ser Leu Val Thr Ser Pro Ala 130 135 140 Ile Gly AlaTyr Leu Gly Arg Val Tyr Gly Asp Ser Leu Val Val Val 145 150 155 160 LeuAla Thr Ala Ile Ala Leu Leu Asp Ile Cys Phe Ile Leu Val Ala 165 170 175Val Pro Glu Ser Leu Pro Glu Lys Met Arg Pro Ala Ser Trp Gly Ala 180 185190 Pro Ile Ser Trp Glu Gln Ala Asp Pro Phe Ala Ser Leu Lys Lys Val 195200 205 Gly Gln Asp Ser Ile Val Leu Leu Ile Cys Ile Thr Val Phe Leu Ser210 215 220 Tyr Leu Pro Glu Ala Gly Gln Tyr Ser Ser Phe Phe Leu Tyr LeuArg 225 230 235 240 Gln Ile Met Lys Phe Ser Pro Glu Ser Val Ala Ala PheIle Ala Val 245 250 255 Leu Gly Ile Leu Ser Ile Ile Ala Gln Thr Ile ValLeu Ser Leu Leu 260 265 270 Met Arg Ser Ile Gly Asn Lys Asn Thr Ile LeuLeu Gly Leu Gly Phe 275 280 285 Gln Ile Leu Gln Leu Ala Trp Tyr Gly PheGly Ser Glu Pro Trp Met 290 295 300 Met Trp Ala Ala Gly Ala Val Ala AlaMet Ser Ser Ile Thr Phe Pro 305 310 315 320 Ala Val Ser Ala Leu Val SerArg Thr Ala Asp Ala Asp Gln Gln Gly 325 330 335 Val Val Gln Gly Met IleThr Gly Ile Arg Gly Leu Cys Asn Gly Leu 340 345 350 Gly Pro Ala Leu TyrGly Phe Ile Phe Tyr Ile Phe His Val Glu Leu 355 360 365 Lys Glu Leu ProIle Thr Gly Thr Asp Leu Gly Thr Asn Thr Ser Pro 370 375 380 Gln His HisPhe Glu Gln Asn Ser Ile Ile Pro Gly Pro Pro Phe Leu 385 390 395 400 PheGly Ala Cys Ser Val Leu Leu Ala Leu Leu Val Ala Leu Phe Ile 405 410 415Pro Glu His Thr Asn Leu Ser Leu Arg Ser Ser Ser Trp Arg Lys His 420 425430 Cys Gly Ser His Ser His Pro His Asn Thr Gln Ala Pro Gly Glu Ala 435440 445 Lys Glu Pro Leu Leu Gln Asp Thr Asn Val 450 455 5 49984 DNA Homosapiens 5 agtcatactg tattttttac ttgtattttt gttgttttgt gggatttaaaaaatattttt 60 attctgagga tagttgaatc cacaggatac tgagggccag ctgtattcacaacccaaatc 120 acatacaaag cgacaagttc atacacaata ggcctattag aacaggactgttctctcttg 180 tttatcattg cagcctttct agcacaaagc ctgggacatt ctggacatttagtatgtgtt 240 aaatttctct tactacatta tttccaacag tatttactgc aatctgcaattaccttcctt 300 ttgttttgta actgtgtccc ccactagaat gtaagctctg tgcagatagtgtctcattta 360 ttgatgtatc cctggcatct aataaaacac tgacaacaca agcacccagtaaatattttt 420 tgaatgactg aacaataacc agttcataag gctgataaaa ttggtatagctagatgaagt 480 atgattttga gggactatga aaatcaaagt aaccacacaa taaattatcagccctctact 540 tccattcaaa acaagctcct gggaattgaa ttatgaaatc tatcatattactttctctaa 600 agaacttcaa gttgggtgtc aactaaaaag ttgcaggcga ggcgcggtggctcaaacctg 660 taatcccagc actttggtag aactgagtat ctcttgaggc caggtttgaaaccagcctgg 720 tcaacataac cagactctgt ctttacaaaa gaaaaattaa aattagccaggcatggtggt 780 gtgcatttgt agtcccagat acttgagacg ctgaggcaga aggatcgtttcggaagaggc 840 tgcaggaggc catgatggca ccactgcact ccagcctggg tgacagagtgagaccctgcc 900 tcagaaaata ataataggcc acgcatggtg gctcacacct gtaatcccagcactttggga 960 ggctggggcg ggaacatcac ctcaggtaag gagttcaagc ctggccaacacggtgaaatt 1020 ccatctctac taaaaataca aaaaaaatta gccaggcatg gtagtggggacctgtaatcc 1080 cagctactcg ggaggctgag gcaggagaat cacttgaacc tgggagctggaagttgcagt 1140 gagccaagtt ggcactattg cactgcagcc tgggcaacaa gagcaaaactctgtctcaaa 1200 aaataaataa taaaaaaagt ttcaaaatga gaatatatgt ttcaaaacaagtataatgaa 1260 tatacttatt gattggaaaa tataattaga agtatctatc aggctataaattgcttttct 1320 tctcccttcc atggaaatta gttttttttt ccatttttag tcagtatgaaaatacaagga 1380 aaaggaaatt caatcaaatt tactttttaa cattttattt ggaaataatttcaaacttac 1440 agaaaagttg caaaaacagt acaaagaact catacattca tttactgtttttccttttac 1500 cctatatatt agttatttat agctgtgtaa caaataaccc caaagcttagtggcttacac 1560 caagtacttt tcatcttaca ctgtttctga gtcaggattc caggagtggctaagctaggt 1620 ggtcctacct ctgggtctct catgaagttg tagtcagcca aaggcttgaccaaggttgga 1680 ggatctactt ccaaagtgac tcactccgtg gcatttggta ggaggctacaaacagttcct 1740 ggacaactgg atctctccat aggctgcttg agtgtcctga aaacacggaagcaggcttcc 1800 ccaggctcca agccccaaaa tgaatgaaaa agagacccgc aaaggaagatgcagtgcctt 1860 ttatgaccta gcctctgaag tcaatactgt cacttctgtt ttgatctatcaagagtcact 1920 aagcctagtc tacactcaag gggaggggaa ttagagtcca cctcttccagggaggaatat 1980 cattgaatct gtgaacatat cttagaacta ccatacctag tttcagtacttttaaacatt 2040 cgccattttg ctttgtccct ctcttttccc cacctacata tacatacacatacatgttac 2100 tccctaacca tctgagagta gggagcatgc ggtgtatccc tatccctcgtgtttttctct 2160 taaggaaaag gatattctat tatacaacac ggtagttatc aatatctaattttaacattg 2220 tgatacttta aagtccactt ccacttgtgt aaattgccct ttctagcaatgttcccatca 2280 aatttatttt taaacaatac agtaaaaacg tagagggcca caaagggtgacatcggtcag 2340 gtaaggtatt ttttttggca gggaataaaa aaggtcctgg gtctagggaggtaaacaagc 2400 gtgagccagc tgagttctag cgggggtccc tgaacaccaa aaggacaagactgtttctga 2460 aacactacat tatctcttaa gttacccatt acttacggaa aatgattttttactgttccc 2520 ttcggttcct gtcttggtta gaacacagct ggagattgtg ttaatagcttaggacgtctg 2580 tttccgtgag caggtaacaa ctttttgaaa caaattccct catctgctgaagaaggggga 2640 caaaaacggc ccctatcgcc cagaaccgtt gcgaggattt agctagctggtgacgccgga 2700 gcacgaagtt gtacaggtag ccagcagcac ccacgcgagc ccgcggttaccctggccgcg 2760 cggctactgt agagtgggct ggcggcgagc gggcggggcg gtatcacgcgggaggggcgg 2820 ggcccgctcg tcggctgatc gcacgattgt gacgcgccgc cggaggcaggccgggccctc 2880 aagatggcgg cgggcgccca gagcggctcg gcccggcagt agtggtgggacggcactagc 2940 tgctggggcc tgccgccccg ggagtggctg cagcagcgcc aggaatcgaggatggtaaaa 3000 tgacccaggg gaagaagaag aaacgggccg cgaaccgcag tatcatgctggccaagaaga 3060 tcatcattaa ggacggaggc acggtgagct gagttccgcg ccggcgagcgtccctcgggg 3120 cccccatccg gtctctcctt cagaccccca cactgccgtc tctaggcgtcccggtgcctc 3180 cctcccttcc cccaccctgt ccgagctgcc ggtgcctcgg ggtcgcggacccgcatgccg 3240 ccgctccggg aatcgtcctc cgctgctcgg gcttgcggcc tccggggcccgtcctctttc 3300 tttcccgcac ctgccgccct ctgctctggc cgcctctgca ggccctgcggcctcgaaccc 3360 cacgtgcgcc tccgccgcgg ggaggaatgt gcggggctcc cccggcggcccgcccgccgc 3420 gcccctcgtc gccgcagcct cgcctcgcct tcgccgccag gccccgcggagccgtcgccg 3480 cgcttgtcaa ggggctggga accatccctg ctctcccata tgttgctaacggggtggcgt 3540 ctggcgcggg gatcccgctg cggccccgta gtacgttcgc tttctgtttccacgtctctc 3600 tgcgtcggtg ctccggctct gggctgctta cagtaaaccc tgaccggagatgggcttccc 3660 tcacttcccg gagtcggaag catgacggca gacacctggg gcctacattcgaacctgcta 3720 gttttcaaag aaaagtcatc actgtgtgtc ttaagatcaa aagtattagaatcagtcatg 3780 gcctaaggat cggaggagga cactttgaag ggaagaaagg tttgctttttagaaacagtt 3840 gtcatcacag taaactttat gcagtgtgta gttaaccagc tggggacgtaggatttttaa 3900 ttgaaaaaca aaacaaaaca aaactgtttt atgctaacat ttctccgttgctacactgtg 3960 tggtctttgt tgcatccgct gataccgcgt tctgaaatag aatggaaaggtgatatatat 4020 gtttcactta cctgaagtgt gcagaaattg taccattaat tccatttctgtttatatctt 4080 attggagccg cgatcaactg ctagcacagt agtaaatgtg taagtaggccaccattgagg 4140 atttgctgaa ttcagttgaa aaacgtgaca aaattttatg acatttcagaacacggccca 4200 gtcaatatgc caaagtttag aaaacttgag acatatgtaa tgactttggaatatattttt 4260 agtttaacgt ttattatatg ttatagcttt gacatttatt gaaaaaaagaaacaaattcc 4320 tcaagttctt tttattgaac ttgattaatt aaaacattac tttgattagatcggttatga 4380 agagtcatag ctcttttgac caagtaggta agaactatgt ggggagaaaaatactgttgc 4440 ctttgtctac ctttagaaag agacaatatt ttacattctt cataaaatctacaaaatagt 4500 ggcaatgaaa gattgtattt tgtaagacca agtgatattt aagatcagtattttttacaa 4560 aatgtaagaa tgaaactgat taagaaacac agcttccttt tccttggaaagttcagtttt 4620 attacctttc tttggggttt tgtttgattt gctttacagc agatgctttctttccaaatc 4680 ctgtgagttt tggaaaagat cgtttttaaa ctttcttgtc ctattattaaggttgtaatt 4740 aattcttagc ctgctttggg acacaaaata aaatgtttgc accagcaataggtttcacat 4800 agaacaaatg aagacttttc ttgagggctg tgaacatggg ggctattatcatttctcatc 4860 tttatacact taatatttca ttctctattc taagagcact gggcactcctttagaaaagg 4920 ggctttgttt tgtatgtttg gatcccacag ggcctagtat gtgaattttaaagtgataaa 4980 aacacttcta ttttgtacta gcacattcct agatgaattt ttattgtaattttgtttatt 5040 cttatacgta atcagaggat atatttcaat aaatatcagg ggaatattttgcattatttg 5100 tattttaatc catcccagct ttaaatttaa aaagtataac tattgcagtcatagaaatga 5160 ttgtaaaatg gtagttgctt atctacctct ctacttacaa tagttcagactactattatg 5220 aacttttttt gtttgtttgt ttgagatgga gtctcactct gttgcccaggctggaggagt 5280 gcagtggcag gatctcggct cactgtaacc accgcctcct gggttcaagtgattctcctg 5340 cctcagcctc ccgagtagct gggactacag gcacgtgcca ccatgcctggctaatttttt 5400 atattttcag tagagacaaa gtttcaccat attggtcagg ctggtcttgaactcctgacc 5460 tcatgattca cccaccttgg cctcccaaag tgcagggatt acaggtgtgagccaccgtgc 5520 ccagactgaa cattttttaa gaaaggggaa aaaattgcca tttgatactctgttgttgtg 5580 tgttttttaa ttcatcgtat catagaatat ttcagtgcta ttgctgttgacctcagagtt 5640 tcagagtttt tataaagttc cgccaatggg tagattcatt cagtgagatgtctgaggctc 5700 tatggtcggt acatgacagt cgtgaacagt atttcacata cctggtcaatggtactgatt 5760 tgatccccct tctgatttct tcttttcaac aatgttaata aaattctttcccgttgtcct 5820 gctaatgaca tatatgtaag cctatttggc cagtttaaat atttataaacaaaactagta 5880 agagttgtta atgatttttc tgaaaattag agcagattag agcagatttgtagttttcaa 5940 cggctgaaga aataaatcct tctaaatgag ccagattaat cgtaagttactgattttttt 6000 attgaaattg tatttcattg aattgtattt cattcagctg aatgaaaaacaggccaggat 6060 aaagctaaca agtaggctac ctatgtgagt agacacaatt aagataaattacattaaggt 6120 gtgtgatttt atattaggtg tttttaacct gggtctgttc accctgaagttgtttgcaaa 6180 attttctttt ggctatacat gtttcttgga agagtcccaa aaggtccatacttcccaaaa 6240 gtttaagagc aattgttctg tttgaaaaca gcataagtaa ctaaagaataagttccacat 6300 attatattca gtaaatattt aatcatatac tgtatactac ttcactgatgaaagtaacca 6360 tattagtgaa tttgctttta aagcatccat atatagaaat agtttttaggccaggggcag 6420 tggctcacgc ctgtaatccc agcactttgg gaggccagtg tgggcagatcacttgaggcc 6480 aggagtctga gactagcctg gccaacatgg tgaaacccca tctttaccaaaattacaaaa 6540 atgagctagg tgtggtggta tgtgcctgta atcccagcta cccgggaggccgaggcacga 6600 gaatcacttg aacctgggag gccaagattg cagtgagcct agatcacgccactgcattcc 6660 agcctgggtg atggagtgaa actgtctcaa aaaaaaaaaa aaagaagtttttagttacag 6720 gttttcatgt atgtaacatt cagtgtaggt atttaagaca gctgaaataaaaataccttc 6780 tgacattttc aaatactaga attctgtttt gttttattaa agcattaccacttgttttta 6840 agcattcctg ttagaggcta agagctaaag agttatttac agtattcaaattgaattttc 6900 cttatctttt aaaatgctca tcttaaaata tgatctttat tgttttggccatacaattgt 6960 ggaactacat ctctgacagt ggaaaatgta tagttctttc agaagtttgtggtaaaatga 7020 ctttaaagat ttgatagaaa gtaaggcata tctgaattgc atggtcggaagtacctgaaa 7080 aaagtaaaat tgatatatca tttgaaaatg aaatgcatat ccctggataagcagagcacc 7140 agattttttt tttcttggca tccctgattt taattaaata ggagtcagcaaccgtttcaa 7200 gagcaggacc caagctctga ccctttgcac tcttcacctg caaggatggctgaagtagtg 7260 gcaggaaagc tctctgggat gtagggcctt tgtagaccca gagagctgttaaataacctt 7320 tggttgctag catgcaagca ataagaaggg cctgtggtgc ttttctttttctttcttttt 7380 ttttttcttt tgagacagag ttttgctctt gttgctcagg ctggggtgcaatggcgtgat 7440 cttggctcac agcaacctct gcctccctgg ttcaaggaat tctcctaccttagcctcctg 7500 aatagctggg attacaggca tgtgccatca tgcccagcta atttttgtatttttttagta 7560 gagaccggat tttaccatgt tggccaggct ggtctcgaac tcttgacttcgggtgatcca 7620 cctgcctcag tcttccaaag tgggattaca ggtgtcagcc actgcgcctggccccgtggt 7680 gcttttcaaa aagcctagaa acatcagggt gtttatattg tctttggcaggtgtgtggct 7740 ggcagcatca ttaattactt agctccttac ctccatggtt cagtgtttggtttagattgg 7800 tgtgtttggg gataaattaa tatgcagttt ttttttcaga tggctatatgcatccagttc 7860 atcctcatgt agttagaaga cttgcatacc aacataatca gaccgtctgcagaaattctc 7920 ctacagttga aatgtaactc ctttgcagct actgaaagtt taaagtttaagtaaaaaaat 7980 gaatagcttt cttcaggtaa cattctgaca agtctgtatg atttaaaagtttcaattata 8040 aggaactctg attgtctttt agcattattt taaattggaa gtgtgaaagtaacagttgac 8100 agtttcagcc agggtacatc aagaagagat gaatatgggt ataatatagctctcaaaatt 8160 tccagtactt tataacaaag aaatatccct cccactgccc tgttttttaaaaaataaata 8220 atacatgttt tccttccagt cgtgggaaac ttaatagaat ggttcaggagggacaagtat 8280 atgcagcata cctgtcattt tccattcaag ttttacttta tttttaaaatttattatttt 8340 ttaaaatatt tcaatagttt tggggtacag gtgggttttt ggttacatagatgtttttta 8400 gtgatgattt ctgagatttt agtgcacctg tcacctgacc agtgtatactgtacccaata 8460 tatagtcttt tatccctctc aagcttcccc cccatcctca aagtccattctattagtctt 8520 acgcctttgc gtcctcatag ctgaactctc acttgtaagt gagaacatacgacatttggt 8580 tttccattcc tgaattactc acttagaata atggcctcca attccatccaagtttctgca 8640 aaagacatta tttcattcct ttttatggct aagtattcaa tggtatatatacaccacatt 8700 ttctttatcc acttgttggt cattgggcac ttgggttggt tccatatctttgcagttgtg 8760 aattgtgctg ctataaacat gcatgtacat gtgtcttttt catataatgacttcttttac 8820 tttgggtggg tacccagtag tgggattgct ggatcaaaca gtagttctatttttagttct 8880 ttaaggaatc gccatactgt tttccatagt ggttgtacta gtttacattcccaacagcag 8940 tgtcaaagtg ttcatttgtc accacatcca caccatctat tattttttgatttttaaatt 9000 atggccattc ttgcaggagt aaatgatatc tcattgtggt tttaatttgcatttccctga 9060 taattggtga tgttgagcat cttttcatat gtttgttggc ttattgtatgccttttgaaa 9120 aatgtctatt catgtctttt gcctactttt gatgggattg tttgttttttttcttgctga 9180 tttgagttcc ttgtagattc tgggtactag tcctttgtca gatgcacagttcataaatat 9240 tttctccaac tgtatgggtt gtctgtttac tctgctgatt tttttttttttttttttttg 9300 agatggaatt ttgctcttgt ttcccaggct ggagtgcaat ggcatgatcttggctccctg 9360 caacctctgc ctctcaggtt caagccattc tcctgcctca gcctcccaagtagctgggat 9420 tacaggcaca caccaccatg cctggctaac ttttttgtat ttttagtagagacgagtttt 9480 ctctatgttg gccaggctgg actcaaacta ctgaccttag gtgatccacccgccttggcc 9540 tcccaagatg ctgggattac aggcatgcct aggcggctat aagtattttgctttatttct 9600 gggttatctg ttgtgttcca ttggtcttca tgcctatttt tataccagtaccatgcggtt 9660 ttggtaactg tagccttttg tataatttaa agtcgggtaa tgtgatgcctccagatttgt 9720 tttttgctta gtcttgcttt ggctatgtgg gctctttttt ggttccatatgaattttagg 9780 attgtttttt cttgttctgt gaagtatgat gctggtattt tgatgggaattgcattgaat 9840 ctatagattg ttttggtcag tatagtcatt ttcacaatgt tgattcttcccttccatgaa 9900 catgggatgt gtttcccttt gtgtcattta tgatttcttt taacagtgttttgtactttt 9960 ccttgtaaag atctttcact tccttggtta agtgtattcc taggtgttttgttttttttg 10020 cagctattgt aaaagggatt gagttcttga tttgattctc agctttgtcgttgctggaat 10080 atagcagtgc tattgatttg tgtcattgat tttgtatcct gagactttactgaatcgttt 10140 atcagatctc ggagcttttt ggatgcgtca ttagggtttt ctaggtatacagtcatatca 10200 ttggcaaaca gtggcagttt gatttcctct tttccaattt gcatgctcgttattcctttc 10260 tcttgtctga ttactctggt taggacttct aaatttttta attactatgggtacaaagta 10320 gatacagata tttatcaggt acatctgata ttttgataca agcatatgttgatacaggta 10380 tacagtgtat aataaatcag ggatactggg gtatccatta cctcaaacttttatcatttc 10440 tttgtgttag gaacatgcca attccacttt tattttattt tattttttattttttgagac 10500 agagtctcgc tctgtcgccc aggcgacata catagtacag tagtgtactccagcctgggt 10560 gacggggaga ctctgtctca aaataaataa ataaataaat aaataaatctgttcagacta 10620 atgtcctaga gtgtattccc aatgttttct tctagtcgtt tgtggtttcaggttttagat 10680 ttaagtcttt aatccatttt gatttgattg ttgtacatgg caagaggtaggggtataatt 10740 ttattcttct gtatatggat atccactttt cctagcacca tttaggagactatccttttc 10800 ccaatgtata cttcggtgcc gttgtcaaaa atgagttgac tgtaaatgcatggatttatt 10860 tctgggttct ctattgtgct ctattgtcta tgtatctgtt tttataccagtattatgctg 10920 ttttggttac tatcactttg tagtataatt tgaagtgaag taatgtgattcctccaagcc 10980 tcggtttttt tttttttttt tttttttttt tttttatgag acagagtctagctctgtcgc 11040 ctaggctgga gtgcagtggc gcaatcttgg ctcactgcaa cctctgcctccctggttcaa 11100 gtgattctcc tgcctcagtt tcccgaggaa ctgggattac aggtcccaccaccacgcctg 11160 gctaattttt gtatttttag tagagacggg gtttcacttt gatggccaggctggtcttga 11220 actcctgacc tcaggtgatc cgcccgcctt ggcctcccaa agtgctgggattacaggcgt 11280 gagtcactgt gcccagcctc cagccttgtt ctttttgctc aggattgctttggctgttct 11340 ggctcttgtg gttccatata agttttagga tttaaaagaa aaaattctgtgaggaatgtc 11400 atttgtagtt tgatagtaac tgcattgaat ctgtagattg cttttggtagtattaaaatt 11460 ttaacagtat tgattcttcc aatttatgaa catgaaatat cttcccatttgtgtgtgtgt 11520 cctcttcaat tcgtgtcatc aatgttttgt agtctgtaga catctttcacttctttaagt 11580 ttattcttag gtattacatc tgtagctatt gtaagtggga ttattttcttggtttctttt 11640 tcagatattt gctgttggca tatagaaatg gtactgattt ttgtatcctgcaacttcagt 11700 gaatttgctt ccattctgat agttttttgg tggagtattt agggttctctctatataagg 11760 tcatgtcatc tgtaaagagg gacagttttg acttcctgtt ttctaatttgcatgcctttt 11820 atttcttact catgcttaat tgctctagtt ggtactttcc agtactttgttgaataagag 11880 tggcgaaagt gggcatcttt gtcttgttcc agatctttga ggaaaggctttcaggttttc 11940 cctgttcagc atgatagctc tgtgtctgtc atatatggct tttatcatattgaggtatgt 12000 tccttctata ccatttttga gagtttttat gaagcagtgt tgaattttagtaaatgcttt 12060 ttcatcatta attgaaatga tcattttatt ttccttcatt cttttgaaatgatgtatcac 12120 cttgatagat ttatgtatgt tggactatcc tttcatacct ggatgaatcccacttgaaca 12180 tgatgaatga tttttttgtt tttaattttt ttgagacgga gttttgctcttgttgcccag 12240 gctggaatgc aatggcgcaa tcttggctca ccgcaacttc cgcctcccgcgttcaagcga 12300 ttctcctgcc tcagcttcct gagtagctgg gattacaggc atgcgccaccacgcctggct 12360 aattttgtat ttttagtgga gacggggttt cttcatgttg gtcaggctggtcttgaactc 12420 ctgacctcag gtgatccacc cgctttggcc tcccaaagtg ctggaattacaggtgagagc 12480 cactgcgccc ggccgatgaa tgatcttttt taagacctcc ttcctgaaggaggtttgcta 12540 gtattttgtt gaggattttt gcatcaatgt tcatcagaga tattgtcccatagtttattt 12600 tgtttttctc catgctagtt ttaggtaatt tttctcttaa ataaacaaagcattttcctc 12660 ctaaagtgca agcatgctta ttagaaaaga tatggaaaat tcagaatagcatagtaaaca 12720 atgtgatatc acttaaaatc attacctaat ataaatttta tttacattgaggtcagtatt 12780 tattgttttt cagagttgaa attaccctac ctatacatgt tatatcctactttgattttt 12840 aaaaaaatta gcatgcttta agccctgaga agttgtacca agctttctgctaggggctgg 12900 gtatatgtgg tggtgaacat ggtggacaaa acaggtttaa agcttatcaaatttgtggcc 12960 aatttttttt tttttttcag agtctctgtc gcccaggctg gggtgcagtggtgggatctc 13020 aactcactgc aacctctgcc tcccaggttc aagcgattct cctgcctcagcctttctgag 13080 tagctgggat tacaggcaca cgccaccatg cccagctaat ttttgtatttttagtagaga 13140 cagggttttg ccatgttggc aaggctggtc tcgaactctt gacctcaagtgatctgccca 13200 ccttggcctc ccaaagtgct gagattacag gcatgagcca ccatgcctggcctcctgtgg 13260 tctttttttg accttatatt actatgcttg ttccatttga tactaggcatcacctcctcc 13320 ttcctgaaac tgctccattg tcatatgtga cactgtattc tcttcactctcctgatattt 13380 tcttactgtt ccttttatct tcctcttttc tttatcaaag aagaaaaaccttcactattt 13440 tcctgtgcct cctacttaaa atgttggcgc ttcttggggt tctgtctcagcccactgctg 13500 ttttcacact tgacactcct gataatctca tctactctgg tggtttcagatatcacattt 13560 gctgctaatt gttttaatca gtgctcaaag acaatacaaa tgtttcaagtaaagagggca 13620 gttttgtaga taggacctga agtaaatctg agcctcgtgg ggggaagtgctgggaagcca 13680 ccagctttaa ctgctagaca accaagctaa acacttggaa gttgttcttgattctccctt 13740 ccgctattta tcaagctcct cccaatttca aatcctgaat ccttaatccgttccctcccc 13800 tccaacattc atactgtgcc actgttttat gccctcattt cttgtttgagctgattcaga 13860 tagcttcctt ttagatgcgc tttgcttctc cattttatcc tttaggaaatcaccagagtg 13920 ataatactgc agtgagtctt aagacatctc tggcagcggt ataaacttaattttgtattt 13980 tctttctcat gtatatcaaa ttccaaatct cttacatact ttcgctggggattgttctgc 14040 ttttgagcca tgttgatatc gtgtttatat ttttgccact tgcttcatttatggtttttt 14100 tttttttttt tggttacatc tttgccagaa taatcttaaa actttcatctgattgtgtca 14160 gtcttaatat cttttagtgg ctccccatgg ccttcagaat taaatatagactccttagca 14220 tggaagctgg tctttgagta cctgtagctt gtctttcaat acacccaacgtgcagcccat 14280 gcactggttg tactgaactc gatatatgag acccataatg ccgcaagccttggaagcttt 14340 gtacaggctg agccatcttt tccaccctat acctccgcct gtctaactctgttgtgtcct 14400 ttcagccttc ctcctggaag tctgatattt cccacctccc aagctcccttggactctgta 14460 tgttccaact gcatactgtg cttatgctaa tgaatttcgt tgttgccttgtctgtccctc 14520 tgactttgaa gacagaggca gtgagtacag atgtttgaca cagtgcccagtacatatatg 14580 atcttaatat ttgttgacta ttaacatcgt tgttattgtt aataattatagaatgtactg 14640 ttaacttttt ttaacttttt aaaaaatctt gttttttata gcctcaaggaataggttctc 14700 ctagtgtcta tcatgcagtt atcgtcatct ttttggagtt ttttgcttggggactattga 14760 cagcacccac cttggtggta agtaatcttt taaattattt aacactgactccaaaatctc 14820 ttcttcttca gttttggagg aaaatgtggg ccttttccct ttgcacggttaattctccca 14880 ccagtattgt tcagtattca ccagtatttt actggttgtc ttttccaactgttaactctc 14940 ccttaccttt ttttgggagg ggggtggcgt ggaggtgttt gaatttggacttgtcactgg 15000 gcatgttcaa gcagaggctc tgtaactact ctgagtaaaa tggaagagattcttaaaccg 15060 acaggtttag aaaagatgat gtctgtgacc tgcatgactc ggcataattactttgaggtt 15120 catttatgca gctgtacttt ccaaaaacag gtttctgttc atttgggctaagtacctaga 15180 agggctattc tttaatagat ctaagctgat tttacccaaa ttctcccaggtttgaaactt 15240 tagaaaagac ctccctgccc gaccaaacaa ctcagaagat agccagttttcttatattgg 15300 tgtagataag gggaatggaa ggagggaagg actatctatg gtaaatatctataccatctt 15360 gaaaggagta attatgataa atgtacagtt taccaaatcc tagaggaatagagttttaaa 15420 gtaatatact atgttttcat gaaggttttt ataaaaaagt tatttaatagaaaaattatg 15480 taagtagatt gaactagcct aagaacattt acagtacata tttcttgatatatttattga 15540 cagctgtgta attgttacta tctatacata aaatattgat gtttagcagttgcttatgcc 15600 tgtaatccca gcattttggg aggctgggtg ggcagatcgc ttgagctctggagttgagac 15660 cagcctgggc aacatggtaa aaccttgtct ctacaaaaaa tgcaaaaattagttgtgcat 15720 ggtggcatat gtttgtagtc ccagctactc gggaggctaa ggcaggagaatcacttgagc 15780 ccaggaggca gaggttgtag tgacccgata tcgtgccacc acactccagcctgggcgacg 15840 ggagtgaaac cttgtctcaa aaaaaaaaaa caaaaaaaaa aacagccgggcgcggtggct 15900 cacacctgta atcccagtac tttgggaggc caaggcgggt ggatcacgaggtcaagagat 15960 tgagaccatc ctgaccaaca tggtgaaacc ctgtctctac taaaaatacaaaaattagct 16020 gtgcgtggtg gtacgcacct gtaatcccag ctacttggga ggctgaggcaggagaatctc 16080 ttgaacccgg gaagtggagg ttgcagtgag ccgagactgc accactaccctccagcctgg 16140 atacagggtg agactctgtc tcaaaaataa aaagtcattt tgaatatatagagcatgttc 16200 atgagtattg ctataaaaaa atatcagagg gttttttttt tttttttagtttactgattt 16260 cagatagaaa tctttaaaaa attaatttac acatttcctg gcttcataatccaagtacaa 16320 cgatttggaa cttcctcaga tgatgcaagt tgattatgac attcataacttcattgaatt 16380 gtaataacct gtttttgtca agggttactg aagtgctgta ataactttttgggctcatga 16440 ctttacatta gctttcctaa tgcgccagcg tgctttttat aatctgtcagtttaacatac 16500 aaatctgtct ggtagaccat cactcctacc atttaaagta cttgagctttgtaatagtaa 16560 acagcccacg tgtatttata ttatgatgga tctaggcaca gtcctttaatgtattcaaag 16620 tggactatgt taagcacagt cctaatgtat tccactttga atacattatcattttttcat 16680 ccttacaacc accttatcag tgagatatta gcctcataat acagatgaggaaaccggggc 16740 ttagaaaagt taagcaattt gattgctact ctgacagtaa gctgcagtgttggtatttgc 16800 acccaggctt ccttgactcc tccagtgctc agtcttttgg ggaatgcaggtagtaacttg 16860 tttgtaccca tgttttagat agttgaggtt gtcaggcagc ccaaccactagctaagtagg 16920 gtgatcaaaa tgtggatgag ctgttagcaa gctatgaaaa aaagcattttgtgatgtttc 16980 cataatttgt tatcagtatt tcaagtgtgt atagctattt ttaaaatttgcttcttgttt 17040 aaattttttt aggtatgtta tctttcgtgt tattttggta catttttttcctagttggac 17100 aaagggaggc tatctttttt aagaacaagg aaggagtccc cttaattagaaaggcttgtt 17160 tattcatttt tcatagacta atgtgcttaa tatattcctt tttttttttttttttttttt 17220 tgagacggag tctcgctctg tctgtcccca ggctggagtg cagaggcacgatcttggctc 17280 actgcatccc ccacctccca ggttcaagtg attttcctgc ctcagcctcccaagtagctg 17340 ggactacagg cacatgccac catgcccagc taatttttgt acttttagtagagatggggt 17400 ttcaccatgt tgaccagaat ggtctcgatc tcttaacctc gtgatccgcccgccttggcc 17460 tcccaaagtg ctgggattac aggtgtgagc cactgtgcct ggccaatatattcttattat 17520 ctttaatttt tgttttcttt ttcttttttt ttttattttg tttgtttgtgtattttgaga 17580 tggagtctca ctctgttgcc caggctggag tgcagtggtg caaccttggctcactgcaac 17640 ctccacctcc caggttcaag caattctcct gcctcagcct cccgagtagctgggactata 17700 ggcacgtgcc aacataccag gctaattttt gtatttttaa tagagacggggtttcaccac 17760 attggccagg ctggtcttga actcctgacc tcaggtgatc cgcctgcctcggcctcccag 17820 agtgttggga ttacaggcat aagccaccat gcccagcctg gcatatctactttttagaag 17880 tgaccctgtt atatattcag tatatgtcac taattaagaa caatatattaattcaatatg 17940 ggctttttaa aaaggtttta ctcatttcaa ggctttttgc ttacaaattttgtttttttg 18000 ttgttggctt tgttggcagt ctttgttttg ggccccagta ctcctcccactcctccccag 18060 cattgtgtgt gagaggtgtg taaagaggtg ggtttctggg taaaagaaggctctcctctt 18120 aaaagtctgt taatctttaa acatttcatt tctgttttat gtgttttgaaactgattata 18180 aatggtgcat gccacaagag tcaaagtttt taacattcat tttaaaaggaaaatgaggat 18240 gaagacataa tttaatttat attttaagtc agtatctttc atttccctgtccctccctca 18300 acagttatat catagtttgt ttcagcattt cagatttcaa agatattctttgaagtattt 18360 ttttaatcag ataaccagtt ttagacatat taattttgaa tgtctggtttgggatttatg 18420 atagccttaa tttcttaatt tttaaaacta atgtgacatt ttaagaccaaaaaaactgtg 18480 tgttgcaatt atctttcact tttaagccct catagaacag tcaaaaaacaaaagctgtgt 18540 tttgtggaag atctgcccag gggaagatgg tgagcctcta ccaacaaggggatttagcta 18600 aaaagaagga ttttgtactg acaaatattt ttaaagattg aggtctaacacttttgagag 18660 gttatgaata tatggttggt catagtagat agttcagtca gaatcagtgattattgcttg 18720 attatgtaac atattagcta agtgatgaga ataacagtag gtataaggatctgtaatgcc 18780 aaggagtgga atttaccggt tttttttttt ctttcctttt ttttttttttcattgagacg 18840 gagtcttaat ctggcatcca ggttggagtg cagtggcgtg atctcggctcactgcaacct 18900 ccaccgccaa ggttcaagag attctcctgc ctcagcctcc ccagtagctggaattacagg 18960 tgcatgtcac cacgcccagc taattttttt ttttattatt ttttttgagacagagtttca 19020 ctctgtcgtc taggctggaa ttcagtggca ctatctcggc tcactgcaaccttcgcctcc 19080 caggttcaag cagttctctg cctcagcctc ccaagtatgt gggattacaggcacctgcca 19140 ccatgcctgg ctaatttttt tatttctagt agaggcgggg ttttaccatcttggtcaggc 19200 tggtcttgaa ctccttacct tgtgatccac ccacctaagc ctcccaaagtgctgggatta 19260 caggcgtgag ccactgttcc tggccggctt tacccttttg acagacctatggctctggaa 19320 ataataggcc agtgtttgat ggttcaagct cctagataca cagtccatgttacggaacac 19380 tcaaaatcca ctagcatctc ttctacctag atggtttcgt gtccttggctacagaaacag 19440 ccccaaagcg tttaacattt taaggattat ttactttcaa catttttaaagttaaaaaaa 19500 agttaagatc cataaaattt tttggaaaag tgttacattt tctctgttcacctctaaaga 19560 ccagtgctaa aggatcctga catcaaaaat ctttacaaca ttcgaattacttgttatatt 19620 tgtctgttaa aattttgtta gaaattgtat ggccccaaag gagaaattgctttggagaaa 19680 aaagttaggt agcagaggaa cagtttggaa gggttggggg ttggccagataaagaaaggg 19740 aagaaacatt caaaattgaa aggatgccgt gtataaaata tgaatattggaaagcataga 19800 atatttcaga aacagtgaag cgaacagatt gattggaatg gaatacagcttggcaaagtg 19860 aatcattagt gataagatct agcatagtat aaaacttctt atagacattcataatgtttt 19920 tcattctttc taacaccaaa cctgttcttc atacctagaa agatttggcttgcagtaggc 19980 cctatgtgat tattgaaaga aaagcataat acatttgagt cccgtaaaaagttttgagat 20040 actagtttaa ggagtttaaa tcttatcctt tagcacaagg actgggaaaatatggctaga 20100 ggactagatc ttttttgcaa tttttttttt ttttttgtag ttgcttctggcaactttctc 20160 tttgtgtgtg tgtttatatt ccttttcaca agtatgttga attgaactttttcctaatta 20220 tcacttagct acttagttaa tgcatgcagt agaactctaa aaagaacttctaggagtttc 20280 tcaaagacct cccagtaatt cttttcaatt agagagggca tgccatttttccttttttat 20340 ttttaaataa tattttattt tttatttttg tgggtacata ggtgtatatatttatggggg 20400 tacatgagat attttgatac aggcatacag tatgtaataa ttacgtcagggtaaatgggg 20460 tatccatcat ttcaagcatt tatcctttct ttgtgttata aacaatccaattttaggttt 20520 tgttttgttt tgttttttgt ttttttgaga cggagtcttg ctctgtcaccaggctggagt 20580 gcagtggcgc gatctcagct tactgcaatc tctgcttcct ggattcaagcaattctcctg 20640 cctcagcctc ccaagtagct gggactacag gcacctgcca ccatacccagctaatttttg 20700 tatttttagt agagatgggg tttcaccgta ttggccagga tggtctcaatctcttgacct 20760 tgtgctctgc ccgcctcagc ctcccaaggt gctgggatta caggcgtgagccaccacgcc 20820 tggccaaatt ttagttattt tcaaatgtag aataaatgtt ggctgtagtcaacctgttgt 20880 gcctatcaag tactagatct tatttattct atttttttgt gcccactaaccatcctctct 20940 cccactaccc ttcccggcct ctggtaacca tcattctgct ctgtctccatgagttcagtt 21000 gttgtaattt ttagctctca caaataattg agaacatgtg aagtttctccttttgtgcct 21060 ggcttatttc acttaacata atgacctcca gttccatcca tgttgttgcaaatgacagga 21120 tctcattctt tttctgtgtg tataaataca ttttctttat cctttcattcatctgttgat 21180 ggacatttag gttgcttcca aatcttagct attaagaata gtgctgcatacaaaaattag 21240 ccaggcatgg tggtgcacac cgtaatccca gctactcagg aggctgaggcaggagaattg 21300 cttgaacctg ggaggcggag gttgcagtga gccaagattg caccatcgcactccagcctg 21360 ggcgacaaga gcccaactcc gtctcaaaca aacaaaaaaa ggaatagtgctgcagtaaat 21420 gtaggagtac agctatctct tcaatatact gatttccttt ttttggaggggtatatacct 21480 agtagtgaga ttgctggatc atatggtagc tccattttta ggttttttgaggagccttcc 21540 aactgttttc cttagtgatt gtactaattt acattcccac caacagtgtatgagtgttcc 21600 cttttctcca catccttgcc tatcttttgg ataaaagctg tttttaactggggtgagatg 21660 atatttcact gtagttttga tttgcatttc cccgatgatc agtgatggttgagcattttt 21720 tcatatacct attggtcact ttgagaaatg tctattcaga tcttttgcccgttttttaaa 21780 aatcagatta tgagattctt ttcttacaga attgtttgag ccccttatacatttttgtta 21840 ttaatccctt gtcagatgga tagtttgcag atattttctc ctattctgtgggttgtctct 21900 tcactttgtt gtttgctttg ctgtgcagct ttaaacttga tgtgatctcatttgtccatt 21960 ctcactttgg ctttggctgc ctgtgcttgt ggagtattat caagaaatctttgcccagtc 22020 cagtgtcctg gagatcacat actattttta taaataaaat tttattggaacacagtcaaa 22080 cccattcatt tacatacagt ctgcgactgt tttttttttt cctttttctttctttttttt 22140 tttttttaag acaggctatt actctgttgc tgaggatgga gtgtggtggcacgatctcag 22200 ctcactgcaa cctctgccct ccgggtttaa gcgattcttc tgcctcagcctcctgagtag 22260 cttggattgc aggcgcctgc caccacgcct ggctaatttt tgtatttttagtagagatga 22320 ggtttcgaca tgttggccag gctggtcttg aactcccacc tcaggtgatccatccgcctc 22380 ttccttccaa agtgttggga ttacaggtgt gagccaccac acctggcctctaaattgatt 22440 tttacttaca atgagcacat ttttgttaaa tttctcgcta ttggcaggagaagaataact 22500 gaagaaaggg gagcaattct gatccttcta aaggttcttc ttgcaacatgtcagaaagta 22560 tatttagcat aatgtttctt cttaaaggga agaccttccc taccttccttattacccaca 22620 ttcccattct ctgttgttat tactgagcga tagcattgga taatagaagcattagtttct 22680 aagtcaaaca ggaactcagt tgcctcatat gtaaagtgat aatattatctaattcacagt 22740 gttgggatta aacaggagta catataggct gtaaaaatgg tagctgctgtttatttttcc 22800 agttgcctgg aattgccttt tcatttgatg cattccagcg gttctcttgctgcccactgc 22860 aaaaaattga taccacatga tttgagaaca agccttggaa aggatagaataacttgttat 22920 acattttcat aggttgggat tttttttctt tatagaatct ttctagatctacttcgtggc 22980 aattaaaaat tacttattaa ttttcccaat ctcctatcct agataatatatccatctgaa 23040 agagaattat aagtcagtta ttttggggaa gcagcatagc atagtgagtaaaaacatagg 23100 ctttcaagtc tgatctctta ggttcagctt cagctttgcc atttacttactgactgtaat 23160 cttagacaag atgtttaacc tctgcatatc agttttctga tagggctgttatgagggatt 23220 aaatgagata atatatgtaa agtgcccaat gtagtgccct gtgacatattaagtaccata 23280 taaatatttg gttattaatg gtcatatgca tgtcatacaa atctgaatatgtaaaataaa 23340 tcagattgta gtatgaatgg atgttcaaaa aggtaaatgt agaaattttattaagactga 23400 aatatagcat gtgattttta ttttggtttt tattctttag gtattacatgaaacctttcc 23460 taaacataca tttctgatga acggcttaat tcaaggagta aaggtaggatcagtcataca 23520 tatatatgtg tatatataca tacatacaca tacacgcaca cggatgaacatacataaata 23580 catatatata actatgcgaa tatatgtttg tttactgatt aataacttaatttttataat 23640 tagatggagt atattttgag agataactta atactttcta gacttgagttttaaatatga 23700 tctaactaca atatagccaa tcatgcataa taataaacca catcataacagattccctgt 23760 ttttatgttg gcattttaat tccagagatc tcaatgattt tgtaagactacagattgaag 23820 aggaaaaaac tatactaaaa aacccccatc aatataaaac tgtatcagtagggtaaagag 23880 ttggttatta tatgaattct ttgctctttc tttttcgagt tttttttttttttttttttt 23940 ttttttgaga cagagtcttg cactgtcacc caggctggag tgcaagtggcatgatctcag 24000 ctcactgctg agaacctctg cctcccaggt tcaagcgatt cttctgcctcagcctcctaa 24060 gtagctggga ccacagacat gtgccaccac acccggctaa ttttttgtatttttagtaga 24120 gacagggttt tgccatgttg gccaggctgg tctcgaactc ctgacctaagtgatctgccc 24180 tcgacctccc aaagtgttga gattattggt gcaagtactg tgcccagctcaaattcttta 24240 ctcttgattc agtttgacaa acaagttttc gaaataggta attagcctgttttatatata 24300 tataaatata tgttttatgt tttatatata tatatacaca cacacatacatatacacaca 24360 catacacaaa caggtaatta gcatatggaa ttgctattgt ggatttattgtgattgagaa 24420 tttattagag cagttcatat ttaataccta cctggagccc cacagatgattctaaactat 24480 cttgggaaat tttaaattta tatatagata agcaattgtt tatttaaaagtttgtgatat 24540 atttacttta gaaacaaagt tgttgaaaat ttttctatag gagtaaaatgatttattttt 24600 ggttcatgct catagatgtg tggtattcat ttttttcatt taatttttttagggtttgtt 24660 gtcattcctt agtgccccgc ttattggtgc tctttctgat gtttggggccgaaaatcctt 24720 cttgctgcta acggtgtttt tcacatgtgc cccaattcct ttaatgaagatcagcccatg 24780 gtatgtgcac atttagatta tagcaactaa atatcacttt cagctattgttttcttaatg 24840 ttcctttatt tctttcactt gtgccatctt ggttatgagg ttttaattttatttttctaa 24900 tacatttatt ctttcacaca gataagaagg cctctaaaaa tacagtaggtaatagttcat 24960 aaagatttta gaaatagttg acactgttgg aggcatctag acttctggctaacttaattt 25020 tggattccag aacccaaatt tcaaaacaat attttgggga ctggataggattgctaactt 25080 tttttcttgt atagaatgtt aaaaacagac acaaaatttg tcattatctatattagttag 25140 gaataggttt tgctatatat caaaacccaa aataagagtt gcttaaaaatcaaatttctc 25200 ttttcatgtg aaaaagggtc tgcaagttga gcagtctgga gctggtatggcagttccagt 25260 ggagcagact ttttctatct tgctgttctt ccatcctcat ctctcacctcactgaacaaa 25320 gtggcttctt gagctctagc catcaagtca tactactgat agcaaggtggaaggagttgc 25380 taagaagagc acatcacctt cttttaagga aactttacag aattcccatcccacctctgc 25440 ttgcatttct ttggtcagat ttcagggata tgctgctcct agttacaagagtctggactc 25500 aaggccacct tgattaaaat cagtattttt tttttttcca ctcaggaggcaagggaagga 25560 aagctaacaa aaagggaata attgtcctaa agtcagagcc caatttggttgtagatttat 25620 agatttattc tgactaatgt tctttttact ccactgagta ttaatgttagttctacatca 25680 cggatgggct ttatttccat ggtgttatct acaaaggccc aaaggtttatatggggcagg 25740 atcttcttct tgtagatgag atacaattct aatagaccaa gcctttgatgaaggccactc 25800 atgcacatga aatcttacca aaaattgaac atggaatgaa caaagaatttgctgtgttaa 25860 gcagtgtgtt agaaaaattt tagtagtgat agtcttggtg gaatgatattttgaaagctc 25920 attatgatta tatgtgattt tcagaaacta aacatcagta ttacaatagaaacttcttat 25980 tcccagctat tttggaatta tttatagcaa aatatagttt actctttaaatattttgttc 26040 actgtttata gtaactgtct attctcagtt ttatgaacac tgaatcctgccatataggtt 26100 ttttgtgtgt aaaactgagt tatttgtttt gccagcattt gaaaagctaaagataactta 26160 tggaaaacat ttagttacta tatagaggct caaaataaat atgaagaaacttgttcctca 26220 gtcttgttta ctggattttg ttttttatct agtgttgtgg tgaaaaacagtaatggatgg 26280 attataatag actttagttt gttgctgttt ttggcagaaa aagcaaaatcacttaacagt 26340 tcaacagttg gtcaaaagat ttgcctgaat acatgatcta attttaagtattcctgtata 26400 gtagctgagc tgctattgaa gggctccttc tagccctatg ttttcataatatctgtggct 26460 tatatttatg gaatagttaa tccatgaact atcctagtaa gctgttgactgaaatgagct 26520 gctcttacgc ttaattaact tataaaaatg aaagaagatt aaaacaatggtaattgctct 26580 aaccatttct tgttatcttt cattcctagg tggtactttg ctgttatctctgtttctggg 26640 gtttttgcag tgactttttc tgtggtattt gcatacgtag cagatataacccaagagcat 26700 gaaagaagta tggcttatgg actggtatgt atgtttattc tataccttttgtatctgctg 26760 agaaatgcct tgtttttaag ataaatatta ttataaggag tgcaaacctttgcattacaa 26820 gatttttgcg taaaatatat tttgtaataa aatcattcat tagactacatttaaaatttt 26880 tttgcggtat gaggctatgt aagttttgat tcttttcatt tagtagatattcataagtca 26940 catgtcagaa ttgaaattat agtatatttt accttgtaga gttctttttaacagaatcct 27000 aaaaataaga attatttagt atgtcaagag ttaaaaaaaa tcactactcatttaatgtct 27060 aatctaaaat acaacaggct aacatctagc tcagggatca gcaaaccttttttgtggctt 27120 tgtgggccac gtacagtctc tgtctcattc ttttgttttt gcatgtgtatttatgtttat 27180 aaactcttta aaaatgtaag aaacagccag atttgagcca tagtggtaggtcgccaactc 27240 ctggacactg ttttggtaaa ctaaattatg gcagtataat gtgtcatctatcaaatctag 27300 gaattaaagg aaaaaagcct agtaatagaa tgactactat aggcacaataatagatcact 27360 actgaatagc cagaaatagg acagtgatgc atttcggtaa atgtgagacaaataccttgt 27420 gataaataag gactgaatat tgtgttgggc tgaattagtt ttaaaagggactgatttctg 27480 attcaaagga cgttatagtg aagaatcata agatttttgg ggaggaaacacctatagaga 27540 gaaagttaga aaaagaacta ataatttctg gcctgttcag tggctcacacctgtaatctc 27600 agcactttgg gaggttgagg caggcggatc acttgagatc aggagttcacgaccagcctg 27660 gccaacatgg tgaaaccttg tctctattta aaaaaaaaaa aaaaaaaaaagtgaaaagaa 27720 aaagaactaa tgatttcagt tgtaaacttg gaacattaaa tgatacaaggctgatgatag 27780 ccaggatatt taaaaaatag tctaattaag ctatagttta cataccataaaatttatcct 27840 ttttatgagt atagttcagt gaattttagt aaatttatac tgttatgcaaacaccaccat 27900 aacccaattt ggggttggtc ggttggttgg ttggttcgtt ggtttggtttttttgacgta 27960 atttattttc ccatagccaa agttttgaaa ttaacaattt tcaatctggaggttctgtgt 28020 attaagccat gttctggcaa aaaacaaaac aaaacaaaac aaaacaaaacaaaacaaaaa 28080 acactgaaat cttctagaaa taatatggat gcagaaaaaa ggtggggaagtggccaggca 28140 cagtggcatg tgcctgtaat accaccagtt tgggaggcca aggcagggggattgcttgag 28200 gccaggagtt tgaggctgca gctatgatca tgccactaca ctccagtctagggtacagag 28260 tgagaccctg tctcttaaaa aaaaaagttg gaggggccag gtgcagtggcttataatccc 28320 agcactttgg gaggctgagg caggaggatt gtttgagccc aggagtttgagactagcctg 28380 ggcaacatag tgagacccca tctatacagg aactttaaaa attagccaggtgtggtggtg 28440 tgtgcctgta gtcccagcta cctgggaggc ttaggtgaga ggatcacttgggcctgagag 28500 gttgaggctg cagtgagccg tgatcgcacc actgcgctcc aacatgagccacagagcgag 28560 acctgtctcc aaaaaagggg gttggggggt gcggggtgac ccctgtgatcttttttctga 28620 gcagaaagaa atggctacca agtggagaga actgaggaga agggaaatgacatgaaacaa 28680 ctgtactgac ttgctcactg tgtcacaaat gtgatctctg taaatgccctcaaatgtctt 28740 cagtgaccct catagtgaga accattttcc ctttccccac acttgtgccagagccctgct 28800 gagatctggg tccctctgaa accacaccta gggctgcaat aacaaaataaccactacatt 28860 tgaaaatata tatttatatg tatgtgtgtg tgtgtatgta tgtgtgtgtatatatatata 28920 gtttgttttt tgttgagacg gagtctcgct ctgtcaccca ggctggagtgcagaggtgtg 28980 atcttggctt actgcaacct ccgcctcctg ggttcaaacg attctgctgcctcagcctcc 29040 ccagtagcta tgcccaccac catgcccagc taatttttgt atttttagtagagacggggt 29100 ttcaccatat tggccagtct tgtcttgaac tcctgacctt tggtccgcctgcctcggcct 29160 cccaaagtgt tgggattata ggcgtgagcc atggcgcctg gcccccatgtgaatatatta 29220 aataccattt aaaaaaccac cacaacccag ttatagaaca tttccatcagcccaaaatgt 29280 tccctcagcc ctgtttgcca tctgtcccca tgctccacct gtgaccccaagcaaccaaca 29340 atttagcttc tgtcaccatg gttttgcctt ttctagaaac ttcatagaaattaaataata 29400 caaaacatct tttgtgtcta acttctttca cttggcataa tcttttgagattgatccatg 29460 ttgatactat agatcaatag gttctatttt tgtctctttt ccttttttttttttttgaga 29520 cagggtcctg ctctatcccc caggctggag tgcagtggca tgatcatggctcactgaagc 29580 cttggcttcc tgggctcaag cgatccttct gcctcagcct ccaaagcagttgggaccaca 29640 ggcatgatcc accatgccca gctaattttt ttctttttga gacagggtctcactctattg 29700 cccaggctgg agtgcagtgg tgccgttaca gctcactgca gcctctgtctcccctctacc 29760 tccctgcctc aagtgatcct tccacctcag cctcccgagt agctgggactactaattttt 29820 gtatgttttg tagtgatgga gtttcaccat gttgcccagg atggtctcaaactcttaaac 29880 tcaagtgatc tgcctgtctc agcctcccaa agtgctggaa ttacaggcatgagacactgc 29940 acctggccag tagttttttt tgattgctgt gtagtgtatt cttatccatcagttgatgga 30000 catttgattg atagctagat gtttgaaatt actagaattt tatgtacttgttcaaataat 30060 tgacctttga aaattgaatt gcttgcctta agcaatagag ttgcaagtaagcattcttgt 30120 gaagtttaag ttctccatcc aaaagtcaaa aatggcatag aaacagaataaaattccaac 30180 attaatctct atgctttgaa agaatatggt ccttttcctt tccttcccttcccctttcct 30240 ttcctttgcc cttctcttcc ccctcccctc ccctcccctt tccacttttcactttcactt 30300 tcccctttcc ttttcgcttt cacctttctc ctttcctttc cttttctcttttcccttccc 30360 ttcccttccc ttcccctttt ccctttcctt tcccctcccc ttttccctttcccttctttt 30420 ttctttttct ttccttttcc tttcccttcc cctttcccct tttccttaagccttttccct 30480 aagccttttc ccttttttaa ccctttcctt ccccttttct cttcccctttccccttttcc 30540 tttctcctcc tctcctttcc tttcctcttc tcttctctcc tctcctcttctttccccttt 30600 ctccattcct tttccctttg ctttcctttc ctctttcctt tccagacagggtgttgccca 30660 gactggactc actcttggga tcaagtgatc tcccacctca gcctcctgagtagctgggac 30720 tataggcagg tgccacctca cctgactaag agtgctattt ttatgaagtgtttcctgctg 30780 tcacatctgc taatttgtag gctgttgtcc agtaggctag aaatgtctgcggttaacagg 30840 tttgctctac tcgtgtcctt ttcaacttta atcttcatct tcaccaggcttaaaaaaata 30900 gacttcctca gagttttaga gatgttctta atttatctgt gatttcattcttcctaaccc 30960 tgccaactaa aaagattacc aagctcagtt ttgttccagg gcttaacatattattcatga 31020 gaacaggaac ctccaagtct ttaagcttta tttcagctag cccttcagtatgtatcaaga 31080 taaacgttca tttaatttta atattggaaa agtcacagtg aaattggatttccttagagc 31140 agtggatttt agactccttt cacagagagc acttaagggt ttatggagatgcccttaacc 31200 aagcttgtcc aacccatggt ccacaggctg cacacatggc ccaacagaaattcataaagt 31260 ttcttaaaac attatgcagt ttttttttct ttaagctcat cagctattgttagtgtattt 31320 tatgtgtggc ccaagaccgt tcttccagcg tggcccaggg aagccaaaagattagacacc 31380 cctcccctaa ggaccagcat gactggcagt caaggagggg tgtttgtacagtgcccaggc 31440 tctcaaccct tcctcaacta aaagagttaa aaaatttaaa taggccgggcatggtggctc 31500 acgcctgtaa tcccagcact ttgggaggcc gaggcgggcg gatcacgaggtcaggagatc 31560 gagaccatct tggctaacac gggggaaacc ccatctctac taaaaatacaaaaaattagc 31620 cgggcgaggt ggcgggcgcc tgtagtccca gctattcggg aggctgaggcaggagaatgg 31680 cgtaaacccc gggggcggag cctgcagtga gccgagatcg cgccactgcactccagcctg 31740 ggcgacagag cgagactccg tctcaaaaaa aaaaaaaaaa aaaaaaaaaatttaaataga 31800 ggcagggtct tgctgtgttg cccaggctgg tcacaaactt ctggcttcacgcagtcctcc 31860 caccttggcc tccccaagtg ctgagattac aggcatgaac catcacacccagtcttctta 31920 aaaaaatctc ttttacctat gaatttgcca gtaggattta ttggaacagagggctccaag 31980 gcttagaaag tttgaagaca gtgtcctgag aggctatcat ttattttattttatttttga 32040 gatgcggtct cactctgtca ccctggctgg agtgcagtgg tgctgtcatggctcactgca 32100 acctccgcct ttctggctca aaggaattct gccacctcag cctctgaagtagctgagact 32160 acaggtgcac accagcatgc ccagctaatt tttctttttt cttttttgatacagacaggg 32220 tttctccatg ttgtccaggc tgtttttaag gcaagaatct aattctttacttttcctgcc 32280 aaaggagaga gtataagaaa agtggggcca ggcttggtgt ctcatacctgtactcccagc 32340 ccttcgggag gctgaggtgg gaggatcgct tgagctcagg agttcgagactagcctaggc 32400 aacatagcgt gacttccacc tctataaaaa ataaacaaaa ttagctgggcgtggtggtgt 32460 gtgcctgtag ccccatcagg agatcttcag gcaggaagat ctacttgagcctgagaggtc 32520 aagactacag tgagccgtga tggcaccact gcactccagc ctgggcgacagagcaagacc 32580 cagttccccc actctcgccc ccacaagaaa aaaagataaa tggcacaggtaggaagagaa 32640 aagggagggt gtgcaacaga aggcctgaca taaatcaaga ttatgaaaggagttatgtgg 32700 tgttgaggaa aaaagtagcc tgactaatct ctgtctatcc ttaatttattgcaggtttca 32760 gcaacatttg ctgcaagttt agtcaccagt cctgcaattg gagcttatcttggacgagta 32820 tatggggaca gcttggtggt ggtcttagct acagcaatag ctttgctagatatttgtttt 32880 atccttgttg ctgtgccaga gtcgttgcct gagaaaatgc ggccagcatcctggggagca 32940 cccatttcct gggaacaagc tgaccctttt gcggtaagtt tatactttttccttctcctt 33000 gataaaaaag tgcatgattc agtgcagcat taatattttg ttgtggatatttctttaggg 33060 aaaacatctt gggtttttct tttaacattt tgaaatactt ttcagaatagtttggggaat 33120 atgaaataaa taaaaaggac aactagttgt ccatgagtac actgccaaaaggaatctctg 33180 catattctta agaatagttc agtggttttg atagaaaacc ttatatcaatcagtcttttt 33240 cttgttctag tccttaaaaa aagtcggcca agattccata gtgctgctgatctgcattac 33300 agtgtttctc tcctacctac cggaggcagg ccaatattcc agcttttttttatacctcag 33360 acaggtaaaa tcctcttcca ctaaggtgga cttttctttc attgtctagtgctttaataa 33420 aaatatttaa tcttgagaga actgtaatag aagtggcctt taaaaatgaatatcattgga 33480 cttggtatag tggctcacgc ctgtaatctc agcactttgg taggccaaggtgggtggatc 33540 agctgaggtc aggagatcaa gaccagcctg gccaacatgg caaaatcctatgcttactaa 33600 aaatacaaca actagccagg cgtagtggcg ggcacctgta atcccagctacttgggaggc 33660 tgaggcagga gaatcacttg aacccaggag gcggaggctg cagtgagccaagattgcgcc 33720 attcactcca gcctgggtaa caagagcgaa actccgtctc aaaattaaataaataaataa 33780 ataaataaaa agaatattat ttggtctact agactttacc tcctattctgtgtggctgat 33840 gttccttatg catttctaat gggggttatt tggtatatta agatatttggcttagggaga 33900 aagatagttt tccctgtcca tataggtggt ttgagtttgt tggctatagaattgatggga 33960 tgatttaacc ccttcacctg ctccagcttc tttgtgattt agagcatatgtaagtagagc 34020 agctagccaa aatgagagca aaaacaagta ttttcctcct tgacactagtctcactagac 34080 ggagatcaag cctttaacca atacatgtaa aatgcacaaa atactgcaatatttatttgt 34140 aaaatgattc tgagttcttg ataagtatct ccaatttagt atatccacattgagggaccc 34200 accatggata agtaggcatt tttagtatgt taagatatgt tgtatttcctctgaggaatt 34260 ctttgtttat aaatgaatta catttatttt tttctggccc attaaatgttaatatacaag 34320 tagctgcagt ggttttctat ctggtataca tttgtattca ttaatgttaccttttctgga 34380 aacccttcta gataatgaaa ttttcaccag aaagtgttgc agcgtttatagcagtccttg 34440 gcattctttc cattattgca caggtgagtt tcttttttta gttagagtgatgtcagtgac 34500 cctggctggc catccaaact ggggcctcat ctagtgatgg tatcttggtggaatcaacaa 34560 agtcaggagt ctgaggttga taggttcaga aattcaattt gtttgtgtagctagtgttga 34620 tcagattttt gggactgaca catttaatat aggatatata aacgtaaaagctagtttatt 34680 gatgtatact aggatatatg tctggatgaa gttagaagtt tcatgtcttgtagtccttgt 34740 cctatttatt cactcattca acaaatattt attgaatacc taccgtgtgtcaggcactgg 34800 ggatacagca gtgaacttaa ctaaattctt gcttttgtag agtttacattctggtgggat 34860 aagacagaaa ataaaaacac caatgtaaac atatcagatg tattgagaactacagagaaa 34920 aagctaaggg tcatatggag agtgacttgg gagaatggtc ttaagggcacagggggcatg 34980 gttgagaaga ccttgctaat gcaaggtgac atttcagcag agacgcaaaggtatgaggaa 35040 caagctgtag gattatctgg aagaagagaa ttttaaacag tacaaacaaaattctcagga 35100 gagactgtag ctggcttgtg taagaaacaa cagggggcca gtgcggctccagttgagtga 35160 acagttgaga gcatcttaga aactgaagtc caagaggtag tgagggaccagatcgtgcgg 35220 ggcttacagg tcctcataag acttgagtaa gataggaagt cattggtgggttttgagcag 35280 cagaatgaca tgaaagtgtg tatcttggca gcccagttgg taaacagttgttgtgtgatg 35340 aataagatat taatcaacac aggaatttgg attttctgag gagatatttcatcctggctt 35400 ccaaactgtt tctgtttaac aataagaata ctatcttttt tccagtgaccaccataattc 35460 tctcacatca cagaacattg tcctccttcc tgtgaaaaag ccccaccccctttcttgcta 35520 tttggctttc tgtttctaag acactatgaa gacaatctag tctaagttaatactttttct 35580 caccttagat gtaatctact agattacaac ttagttttat tctagggtatttttaattga 35640 ctcttggatg gttcaccatt tctcgagtag gattccttct gcaggtctcatgattcattc 35700 tgttttggtg agtttagcaa caaatttcaa atttaaatcc tatatgcctccccaagcctc 35760 tgcacataca tatacttggt gttgagtatt agtactattt ctagcttcaggtttgtccta 35820 aaaatcatca gtctggaaaa acaatgcatt taaatattca ttcctagccatgagaaaagt 35880 gctttttaac tttggaggaa aatatactgt agcctttata taaaaatggctttaaaaaaa 35940 gtttttgagg ccaggtgcgg tggttcatgt ctgtattccc agcactttgggtggccaagg 36000 tcagggaccg cttgagccca ggagttcaag accagctcaa gcaacttggcaaaaccccat 36060 ctctaccaaa aaaaaaaaaa aaaaaaaaaa aaagaaagaa agcccggtgtggtggtgtgt 36120 gcctgtagtc ccagctactc aggaagctga gatgggagga ttgcttgatcctgggaggtc 36180 gagggtgcag tgagccacag ttgtcccatg gtactccagt atgggcaacagaatgagacc 36240 ctgtctcaaa aaaaaagtgt aaggaaaata cacagttagt atgtgtagaacttgatgaat 36300 tatcaaagat taacccaacc ttgcaataga tgtgatcaac ctgggcatgagtatcttttt 36360 catacattgc caacattaga tttgctaaca ttttgtttaa gatttagatgaagagagcag 36420 actaatactg taatgacaca taaaagattg atagctataa ggtcttaagttctgtttctt 36480 acttaaatga ccatgggagc tgtatgcatc taataaatgt agtcagtgacactgcagacc 36540 cagtgatgag tggagggtgc ttttgagggt atttttcctc tgtttagcagatggcatctg 36600 gcactaggtt acaagatgaa aaacagtctc tgagagtgca gaatctggcagggcagacag 36660 gtaaaggaga ggaatgtagt tggatttgta ttggaaagat cagtgcagcagcagagccct 36720 gaaggcagta agaccgcaag gtgcaagcaa ccctccaaac accattgctgacacagcatg 36780 acacacacag tccatgtagg aacaaggtcc ttaagtgacc atttaggttttggtgcttat 36840 gtagaagtaa gaattaacac tttatatcat atatgagcta ctacattattacatgtatag 36900 cctcaaaggt agaagaatga tcttgtattc tcatttatgc agaaatatacaattgagaat 36960 gacttgtccc ctgttgacct tccataccac tgaaagacca gtgtatttctccctcctctc 37020 cagcagcacg gagctgctaa tagctctcca ttaatgcatg tttgctttatttcttaccca 37080 cgtgcttttg ttcctgctat tctttctgcc tccccttcct ccaccctaagtagccctttt 37140 ctgggtcccc atgctcatgt gcgcatatct ccatcattgt acagaatacagtgtagtaga 37200 gattttatct ctgtttattg ccttagtttg tgagctctag cggacctctgagtagatgat 37260 gaggtcagga ttatatcata ttcatttttg tcaccctagc accctgaactgccaggaggt 37320 gctaaactaa aggtcattgg tttttttcca taatgttaaa aaaaaaaaaaacttaaaaaa 37380 ttagtagtaa gcactttaaa attgggaagt gttacgtgaa attatagttatgtagcttct 37440 cccccaaaat gattagatct ggtaaccggg tgtgggccaa ccttccagcgagagccaagt 37500 agttgctgtc ccctttggaa ccttttcttt ttggtttatc atcagccccattacttcctg 37560 ggcaccggta cgtataagag ttcctaacac ttgcactaag taagtgtttacatgagaaca 37620 tcaatatagt tctacacatt tcttttttct cagtgttttc ctatccagccctctctgtgg 37680 gggtgactcc cacacttact cttccagtcc agtccctgag ctcctatatgacacattggc 37740 tgggtatctc agccttaaca tggccaaaat taaaatctgg gttccatcccttgcccgcca 37800 cccctatgct cctcatctca ttcagtggct tcaccaccgc caggttttgggggccagaag 37860 ccttagcgac attcatgaat cctttctccc taaccttaca ttcagcccatcaaatgatac 37920 ttcctatcac ctctctctcc aaaatatatc ttgaatcaga gcgtttctgatattctccat 37980 tagtaggaac ccaatctgag ccgtggccat atcttccatc tggtctctctgcttccatct 38040 tgcctcacta tagttcattc tgcacttggc agagtaattt ttgtaaaatggaaatttaat 38100 cgcatcttac ctataactca ctttcctttg caaccagaat aaaatctagactccttatta 38160 tgtcattttc ctccactctc cacatggttg agtatgttct gtttcagtccagggcctttg 38220 cacttgctgt cagccttgct tggggtgttc tctttcccca gatctttgcataactaggtc 38280 tctcccctta gtgagctctc acttcaaaag gccttccctg gatcctggtttaagcagcat 38340 ctccatcaca ctgccctgtt ttattttgtt catagcagct acaacctggaatatcttgtt 38400 aaataagcag gctcatatgc atctttcctc catgagaagg tagtccgtgtgttgatggat 38460 actttgactt actcacccat gcgtcttcag gccaagaata agagttggtcttattaggaa 38520 tttggcaaat atttgttgac agactaacca aagctgatgt tagtgttagcttagctgttc 38580 atcagctatg ccatcttgct taagtcattt aacctttggg actcagtgctgtcatcttca 38640 aaatgagagt taaattaagg gacaccaaaa attttttccg ttcaagaacacttatatatt 38700 aagtattttg ttccattatt attaatatta ttgagaatat tataacatttagaattatgc 38760 atgctttcca taaacactta ttaagaactt actgggtgct ggggatataaatgtaaataa 38820 gagaaaagtt cctgccttca agaagaaaat cagtgttccc agttacagtgttgtaagtat 38880 cggtgtcttt tagggctttg ggtcacaaga taggctgtag gggaaggagtccaagaggag 38940 gttctcacag cagtgggcct gctgcagtaa ttctgcacat aggacgttaccccaggaaag 39000 ggggattggt gtatctcatt gtttccaatt ttaatgactc atctcatggccttaagaaaa 39060 tatattcttg gccgggcacg gtggcccaca cctataacct tagcactttgggaggccaag 39120 gtgggtgaat cacctgaggt caggagttcg agaccagcct ggccaacatggtgaaacctc 39180 atctctacta aaaatgcaaa aaaaatttag ctgggcatgg tggcacgtgcctgtaatccc 39240 agctactcgg gaggctgagg caggagaatc acttgaacct gggaggtggaggttgcagtg 39300 agccagcatt gcgccacagc actccagcct gggcaacaag agcgaaactccatctcgggg 39360 ggaaaaagaa aggctgggca cgtggtggct cacgcctgag ataccagcactttgggaggc 39420 cgaggcagct ggatcacaag gtcagaagtt cgaaaccagc ctggccaatatggtggaacc 39480 ctgtctttac taaaaataca aaaattagca ggcatggtgg tgggcacctgagtcccagct 39540 actcgggagg ctgaggcaga aaaatcgctt gaaccccgga ggcagaggtggcagtgagcc 39600 aagattgtga cactgcactc tagccttggc aacagagcga gactccgtctcaaaagaaaa 39660 aaaaacccaa aagaaaaaga aaatatattc tccagttaat cttatctataaaaaggaaat 39720 gaggctaata atgcattcta agcctttttt attgaattgg gatttattctttgagaacag 39780 ctttccacaa aggggaagat agtcatttct gcagataagt acttactggctagatgggtt 39840 ggttgaaggg ctatgagatg accgcatttt ataagtactt tctggtaatattaatgatct 39900 ctgcttgaga agtgtcagct ttcttagact agcattcctg aaagaacacgtgctccaggg 39960 tacatggctg gcccatgcca tcctgttccc actgagctgg gagttgatgctcagccttct 40020 tcactgtcct gtctcttggc tgaagtgcca gggatttcat cattaagtgaaatctatttt 40080 ttaacagcat ttcttccttg taatgcttat catcatctca atgaatgatgagaacaaatg 40140 ttttctgctc tgtgagttcc tagagccata taaagaatca cagctttttagatgaaagtg 40200 ctaccttccg ggatgttctt aaaagtagtt tcccagaagt tcttcaactttgcattatag 40260 ttccctgttc ttctcagaca gttggaggcc acagagcttt gggtatacttacagtttcct 40320 ttttcataat taattgaaag ccagtctctg atatagtcca caaataaaatcatttttaat 40380 tatttctaac cctaattaga atcctaatca tttctaatct ttctgattattttttataat 40440 attggccacc agttcccacc cacaagtcat gggtgacttg agatggtctctgtcacccag 40500 gctggagtgc agtggtgtga tcatggctta ctgcagcctc gacctcctgggctcgagtga 40560 tcctctggac tcagcctcct gagtagctgg gaccacaggt gtgtgccaccagcctggcta 40620 atttttcaat tttttgtaga gatgtggtct ctttatgttg tccaggctggtctcaaactc 40680 ctgggttcaa gcagtccttc catctcagtt ccccaaagtg ctgggattacagatatgagt 40740 cactgtgcct ggcctaaatg tttctttcag ttgaagtttt tctcagctaactgctggctc 40800 tgggcaagtt tccttttctg gtttggttgc cgaagaatat aattctacatggaactcagt 40860 cttatactca cgtgatttaa gtgaaagtct tagacaagaa agctgtgagttctaattgct 40920 caaaaatcct tgaatgaatc atttgctttt cactggcata tttgtgattaaattcttaag 40980 tggccatctc aatttaaaga attcaaaact tatattttat gagttttaaagtgtcagccc 41040 atcataaaat agtgatttcc tgaattattt ttacttgtct atggacttactagctatctt 41100 atgtagtata ttaaaaacgt tagttagaat agcaaaaaaa aattttctaatgttcccaaa 41160 tcactgctga actttgttga ctttgaaaga aaaaggaggc acaagaaaacccacccactg 41220 ataatattgt ttattaaccg ctgattattg ccaggcacaa ttctaagaactttatgtaaa 41280 tatatctcat ttaattccca tgacaaggtt ttgaaatagg tgctgttatccccatttgca 41340 aagagacaaa agtgaggctc aaagtgaagt gacttgctga gagccacacaaagccaagat 41400 tatgatccag gctgtttttc taaagtctgc tgtatagtat actgcatttatcccaaatca 41460 agcttattaa tttactgttt ataaaaggca tcatggtttc aacaacagattaacttaggt 41520 aaataatata tgggtaatca ttgttctgtt agtttttctt tagttgtggaaataagcatt 41580 ttagactaac ttggacctaa acaagcttta aggctattat gtaatggggatctccaaatc 41640 attagttaga acttttgacc cttccatttt caactactga tttaagtggtcctcagtaga 41700 aatgtactga ataggaagtt ttatctttca gttttctaac tctcagtctggatctatgtc 41760 agcagaggga cttttcatct gcttatgtga cctggactag tgatctcagacatattcagg 41820 gcaattattg ctgaaaatca gccaaattgt agaaaagtgc caatagtccttttatagtgt 41880 agattgaaag aagtcacttt ttaaaacttt attctgataa atctttttttttttttttca 41940 gaccatagtc ttgagtttac ttatgaggtc aattggaaat aagaacaccattttactggg 42000 tctaggattt caaatattac agttggcatg gtatggcttt ggttcagaaccttggtaagt 42060 ataaatattt taatgttaat atttttaatt ttggtgttag cccttgtgtttttatttgct 42120 tctcaactga ggggtagact gtaatctgtc tcatactatg ctttttatctttcaaaatgt 42180 gtctaatata agtctgccac ttgtatattt atatgttctc ctagaatggcttgaggatta 42240 aaaaggtgac cttttatagc taaatgacag gctgaatttt tgaatgagattatacagctt 42300 ttgaatcttt aaggagcatt taatctaaat cagtccgtta ctaggaaaaagtatgtaatc 42360 ccatagcaac aaggccctga aagttattta cattttttgt ttttctggtcaggaaaaaga 42420 aagttgtata accagtgatc attactgata gcaaaccaga agtgaaaacaatccagttat 42480 tttggtgcca gcttcatgct gtgtctcagc ttttctgacc agtcgggtttcctgcagggg 42540 acttgagtag tgacgcttgt tgtcaggctg cccacgtaga tagtattattgtgctcaggc 42600 attatggcac tgaactccat ggtttgcact aatatttcaa acaactgactgcctcgtctc 42660 tgtttggact tggatataag caagcatcaa gagggtgatg atttgttctccaaactagcc 42720 tttgcaaaga ggtgctcaca attgaaatta cctaaaacat ttcttttaaacatcaagcca 42780 ggaacatcca agttacttgt tctttacaat ttaaggatta gatcaaatcagcgatatctt 42840 cacaaatcca tccataagaa ctttgccaaa gacttgttgg cttcatgtgtttggaaatat 42900 ttgatgatgt ttcgtcatct atattataca ttatcctcaa tataacctctcaattgcctg 42960 tagaaataat acccagcaca ttttacagtt tggaacatga tatccctattttacattaca 43020 ccctcacagc actcttgtga attaagttgc tgtctttgta tacagggtcagattgttaag 43080 cgacttgccc atagtcacct agtaagcaag ttcagttctc cttttctctacagccatttc 43140 acagtaagaa ttacttaatt atgtagtttg actttcaggt acagtggagaagaatttact 43200 gtttttgttt tgctgctctc cttataggat gatgtgggct gctggggcagtagcagccat 43260 gtctagcatc acctttcctg ctgtcagtgc acttgtttca cgaactgctgatgctgatca 43320 acagggtgag ttgataggaa ctagcgataa ttatttaaaa gtacagaatgttctaatcct 43380 gtgttctgtc tcctatgtac tgaaacataa gtatatcttc agggtagagacttttaaaat 43440 tgcttttgat ataaacagga aaagcagatt ctagggtatt tatccttaggtagatacata 43500 ttcccttttc tctcacttag aatatgtggc ttatctgttc tgttcatagaaaatttactg 43560 atgaggctgg gcatggtggc tcacacctgt aatcccaaca ctttgagaggccgaggcagg 43620 cagattgctt gagttcagga gttggagacc agcctgggca acatggggaaaccccatcac 43680 taaaatgcaa aaattagctg ggcagggtgg cgcatgcttg tagtcccagttacttgggag 43740 gctgaggttg gaggatcact tgagtctcag aggcggaggt tgcactgagccaaaatcagg 43800 ccagtgaact ccaacctggg cgacacagtg agagaccttg agagacctgtctcaaaaaaa 43860 tttactgatg aatgttggcc acagaatatt aactaagcat taagtttttgctgtgttgtg 43920 ctagacacct tgtgggatat attcatagac cttttatgaa tgttgtcccagccacttggg 43980 aagctgaggc aggaggatta cttgagctca agagtttgaa gctagcttaggcaacacagc 44040 aagactccca tctctaataa aaaaaaaaaa agaaatcata tagtttcagtcatctgaaga 44100 tatgtaacac agcaacatct ttaatgtcat atgctctctt ttttttttttggtgggaaca 44160 tttaattctt gggatgctaa cagatgacaa atacttcttt gaaaaggatatgttttgtct 44220 agtcataagg ataaaagggg cactgcaaaa cagtggcagt tgtcacttggttgataaatg 44280 aggaaggaaa ggcctatggc caaagcaagt tgcattttaa attaaagatccaaaagagag 44340 aaacaaaaat caaatgctgt taaaacagtg ttattggccg ggcgcagtggctcatgcctg 44400 taatctcagt gctttgggag gccaaggctg gtggatcacc taaggtcaggagttcgagat 44460 cagcctggcc aacatggtga aaccctgtct ctactaaaaa aatcaaccaggcgtcgtggt 44520 gcacacctgt aatcccagct actcgggagg ctgaggcagg agaatcacttgaaccctgga 44580 agcggagttt gcagtgagcc gagatcgtgc cactgcactc cagcctgggcaacaagagtg 44640 aaactccgtc tcaaaaaaaa aagtgtgatt ttggctgggc acagtggctcaatgcctgta 44700 atcacagcac tttggtaggc tgaggcagga ggatcacttg agttgagttcaacaccaccc 44760 taggcaacaa agtgagaccc catctctaca aaaagtacaa aaattagccaggtatttgag 44820 cccaggaagt tgaggctgca gtgagccaag tttgcgccac tgcactcccgccagttgacc 44880 cccccacttt atttttgact aaaaggggtc ttaagtactt gcttggggacagataaattt 44940 taacatttgt agttgtgaaa ttgtgactga tttttaacca gccatgttttgaaggctgta 45000 atctagggaa ataaagtagt tttgcccact tgtttctatg tgagacctatatatgggtac 45060 atcagagctc atgtttgcat aggacagctt actacacttt gtagagctgatagcttctaa 45120 atattaatag tttttaatga cattgctata aattgctagt gtctgataagaagcttgatt 45180 ttagattaag tgatttcata tttgtactgg ttctaaggta gggaaaaaaaaccaggtagt 45240 ttactaagtg ataatttgtt taaacaatgt aggtgtcgtt caaggaatgataacaggaat 45300 tcgaggatta tgcaatggtc tgggaccggc cctctatgga ttcattttctacatattcca 45360 tgtggaactt aaagaactgc caataacagg aacagacttg ggaacaaacacaagccctca 45420 gcaccacttt gaacaggtaa ttctcattca acatgatcaa attgtatggttcatttggct 45480 agattaaagg ctactggttt ttggtcatga aagcttttat gtgagattctatttgagatt 45540 ggataaaatg cttaaaaacc agagtttaag ggactgtgtt cttctacataccaccttgtg 45600 aaaattggct gtgcatattt tttttccact tgagaatgaa aaattttaagtacctagttt 45660 gtcaatggca tatgtaacaa accatttctc tttactacta gtctcttttgaaacttttct 45720 aatatcacag ttgtgtatgt taactaactt ttcatacaaa aggcaggcttatggtaaata 45780 acctttgttc actgtttgct atatttccct cttttcaact gaaaattaatgccaaacaat 45840 gctgatcatt ttggccaata ttagcagctc atcagtttcc tggcttatttagcagagttc 45900 tgtacttatc ttccaaccca ctaagactac ttttaaataa aagaatgtttgtgggctata 45960 cacaaaactg gcagtgcaag cttctgctaa gaatgaatgt aattttgggctaaaacaaag 46020 gtctcttttt cttggtaacc tgtgtttttc tcacttccag tcacttgaaattataattac 46080 cttcttgaaa caaagaaatg gtaatttatt ttgctactgt agtaaatactgtatgctacc 46140 tgattttatt ttgttttttg ttgtttctga gacagtctta ctctgtcacacaggctggaa 46200 tgcagtggca caatcttggc tctctgcaac ctctatctcc caagttcaagcgattctttt 46260 aacatcagcc tcccgggtag ctgggattac aggcatatgc caccatgaccggctaatttt 46320 ttttgtattt ttagtagaga cgaggtttca ccatgttggc caggctggtctcgaactcct 46380 gacctcaagt gatctgcctg cctcagcctc ccaaagtgct gggattacaggcttgagcca 46440 acacgtgcag cctgatttta tttttgagat ctttctataa acgttttccccttggactaa 46500 caaattaatc atagaatagc tgtgttcaca ttttgtgctg aaagtaatgatgtaatattt 46560 tcgcataggc tgttttgcca gttgttattc ccagaatttt atagagaatgtgatagtatc 46620 tcttttctcc taaaaaggga atgcctttta taattatggc tttaataaagagaagagcaa 46680 ttagcttgta attcataagt taatactaaa aggtatgtag tttctccttatgagtaaaag 46740 tatttgtgta aaaatcctaa ttactttatt cttcctcaga attccatcatccctggccct 46800 cccttcctat ttggagcctg ttcagtactg ctggctctgc ttgttgccttgtttattccg 46860 gaacatacca atttaagctt aaggtccagc agttggagaa agcactgtggcagtcacagc 46920 catcctcata atacacaagc gccaggagag gccaaagaac ctttactccaggacacaaat 46980 gtgtgacgac tgaaatcagg aagatttttc tatcagcacc caggtcttagttttcacctc 47040 tagttctgga tgtacattcc atttccatcc acagtgtact ttaagattgtcttaagaaat 47100 gtatctgcat gaactccgtg ggaactaaag gaagtgggaa cttagaaccagacagttttc 47160 caaagatgtt acaatttctt ttgaaaaacc ttttgtttat tagcaccaatttcttgccac 47220 taagctattt gttttattat acatccttta attaaaaact atatatgtaacttcttagat 47280 attagcaaat gtctctgcta ccatttcctt aaggtgttga gctttaactctatgctgact 47340 cagtgagaca cagtaggtag tatggttgtg gacctatttg ttttaacattgtaaaatttt 47400 gagtcagatt ttaatattgt aaaatcttgg gtcaaataat tcaaagccttaatgcagatg 47460 cactaaaaca aagaaatggt aaatgaattg tttgcattta aaaaaaaaaactcttaagaa 47520 aactgtacta aatctgaatc atgttttgag cttgtttgca gtacttttaaacattattca 47580 ctactgtttt tgaagtgaga aagtatcagc catttagcat ttaagttggggtatttagag 47640 cctgtaatct aaatgctggc tcaaatttat tccccagcta cttcttataccactattctt 47700 ttaatgtttg cataatcata agcacctcaa cacttgaata cataatctaaaaattatata 47760 gtaaagctgg tagccttgaa aatgtcagtg tgatatctat tatgtagataaatatatata 47820 gtggcctttc aggactgtca cagtaacact ttatttacag agctaatgtttgtcctaaat 47880 tttcaggacc ctagaggaga gctttataca attaccgatg tgaatttctctaaagtgtat 47940 atttttgtgt ccagttatat tatttaaaaa agtgttactt tgtaaaaattgtatataaag 48000 aactgtatag tttacactgt tttcatcttg tgtgtggtta ttgcttaatgctttttaaac 48060 ttggaacact cactatggtt aaataaggtc ttaaaagaaa tgtaaatattctgttaataa 48120 agttaaatat tttaatgatt ttttttttaa aaaagtcttg atctgtgagttcctacaggg 48180 tctgttttgg ctttgttacc atatttttac agtaacaaat caaagcattattcattcttt 48240 ctttgcatcc atttcttttc cctcactaaa aaatgctctt caaggaagacaagaggaaag 48300 aaacaaaaat gcagtatgcg ttttgaagta cttgattttc aaagtgtagatgctgcataa 48360 tagtttatta acaaagctca aagtttacag aaataacatt aaatgcaacactttttgatt 48420 attaacacat gtacaatttt acactgcaaa acaattgcaa ctaaaagtttaggaggtaaa 48480 gaattagcac acttggaagt ctgtatacat ttaatacaaa tttgcaagatatcagaatga 48540 tctctcctga aaattcaact ttccttgtgt atatatatgc aaagagatacctatatttta 48600 aaaaagagag ctacctgtat gtcatgcatc ccatccaaaa catgtcacaatattttatat 48660 atattacata gtatttacaa caaagcccct tccatagtat ttacaataaagctccttcca 48720 aaatcaccaa gaggcttctt tccagaatag caagtgcttt caagttactgtaccaagtat 48780 tctcactaat acagtagtgt atctaaatgg gggaggtggg ggagtatcaactgcctaata 48840 ttagttactg aattttcaga ttagatccct agtttaaaga cacttgcaattgccaatagt 48900 ttcaagatac tggcttcgta aaaaaaaaaa aaaaattagc aaagattctttcttaccatg 48960 ggactcagta atgttattac atcaatatct tgaaatgttt cttcatctgtgtaataaagt 49020 agttgaaaat aattttaaat tatcacatga gcattagtac tttataaaaattgatctaga 49080 agactcttta gagaatgcta ccaggtattg tttgtcaata agaaaacaaaatgaggccct 49140 atgattctag gtgaatttta aaaaaatttt tccccaagga acaattcttaaaactttcag 49200 ttacagggaa gagaagaaaa ttgtaccttc taacagttat ttgttttgcctagtttatta 49260 acattgaaat caacccaagt tctgatacta taaaaataaa tgaatattcatgatatagta 49320 gatttagtag tagtaccaag ttttaaaagt ccactgatac agatttaactgcataataaa 49380 actacaaatt gagaaaaact agatgagtat aaacatttag gaagcactgaagttttaaaa 49440 acttgtcagc tcaaatgaca aaagcactta atctggtcac ataaaaactgtccaaattat 49500 tttaagtata tcaaatttat ttgattcatc actagcaaat ttaaatgcttcaaggaaaat 49560 acgctatgaa ggcttaattc atttcagtta tttaaggtaa atttaggacttttcccttta 49620 aatgtcatca ataatatatc tcaagaggta atcattttcc ctcatgattttcaggtgttg 49680 aatttaaaag ttttaccaat gaaagtgata aaatgaatta gtattctttggttgcatact 49740 atgaggttat gagcaggttt tagtttaccc agatttttaa tatgatcagaatctccctca 49800 taaggatcaa ctctttcctt agaactgaat ttctaaagaa tagcttaataaaattcatat 49860 tattctataa aacgccatgt tcacaaacca aaaaatgtca tttactcacaatctttacca 49920 atccccaagt acaaatttgt tcctatatta taaactgcca taaaagcagtacttaaagta 49980 tcca 49984 6 490 PRT MUS MUSCULUS 6 Met Thr Gln Gly LysLys Lys Lys Arg Ala Ala Asn Arg Ser Ile Met 1 5 10 15 Leu Ala Lys LysIle Ile Ile Lys Asp Gly Gly Thr Pro Gln Gly Ile 20 25 30 Gly Ser Pro SerVal Tyr His Ala Val Ile Val Ile Phe Leu Glu Phe 35 40 45 Phe Ala Trp GlyLeu Leu Thr Ala Pro Thr Leu Val Val Leu His Glu 50 55 60 Thr Phe Pro LysHis Thr Phe Leu Met Asn Gly Leu Ile Gln Gly Val 65 70 75 80 Lys Gly LeuLeu Ser Phe Leu Ser Ala Pro Leu Ile Gly Ala Leu Ser 85 90 95 Asp Val TrpGly Arg Lys Ser Phe Leu Leu Leu Thr Val Phe Phe Thr 100 105 110 Cys AlaPro Ile Pro Leu Met Lys Ile Ser Pro Trp Trp Tyr Phe Ala 115 120 125 ValIle Ser Val Ser Gly Val Phe Ala Val Thr Phe Ser Val Val Phe 130 135 140Ala Tyr Val Ala Asp Ile Thr Gln Glu His Glu Arg Ser Met Ala Tyr 145 150155 160 Gly Leu Val Ser Ala Thr Phe Ala Ala Ser Leu Val Thr Ser Pro Ala165 170 175 Ile Gly Ala Tyr Leu Gly Gln Met Tyr Gly Asp Ser Leu Val ValVal 180 185 190 Leu Ala Thr Ala Ile Ala Leu Leu Asp Ile Cys Phe Ile LeuVal Ala 195 200 205 Val Pro Glu Ser Leu Pro Glu Lys Met Arg Pro Ala SerTrp Gly Ala 210 215 220 Pro Ile Ser Trp Glu Gln Ala Asp Pro Phe Ala SerLeu Lys Lys Val 225 230 235 240 Gly Gln Asp Ser Ile Val Leu Leu Ile CysIle Thr Val Phe Leu Ser 245 250 255 Tyr Leu Pro Glu Ala Gly Gln Tyr SerSer Phe Phe Leu Tyr Leu Lys 260 265 270 Gln Ile Met Lys Phe Ser Pro GluSer Val Ala Ala Phe Ile Ala Val 275 280 285 Leu Gly Ile Leu Ser Ile IleAla Gln Thr Ile Val Leu Ser Leu Leu 290 295 300 Met Arg Ser Ile Gly AsnLys Asn Thr Ile Leu Leu Gly Leu Gly Phe 305 310 315 320 Gln Ile Leu GlnLeu Ala Trp Tyr Gly Phe Gly Ser Glu Pro Trp Met 325 330 335 Met Trp AlaAla Gly Ala Val Ala Ala Met Ser Ser Ile Thr Phe Pro 340 345 350 Ala ValSer Ala Leu Val Ser Arg Thr Ala Asp Ala Asp Gln Gln Gly 355 360 365 ValVal Gln Gly Met Ile Thr Gly Ile Arg Gly Leu Cys Asn Gly Leu 370 375 380Gly Pro Ala Leu Tyr Gly Phe Ile Phe Tyr Ile Phe His Val Glu Leu 385 390395 400 Lys Glu Leu Pro Ile Thr Gly Thr Asp Leu Gly Thr Asn Thr Ser Pro405 410 415 Gln His His Phe Glu Gln Asn Ser Ile Ile Pro Gly Pro Pro PheLeu 420 425 430 Phe Gly Ala Cys Ser Val Leu Leu Ala Leu Leu Val Ala LeuPhe Ile 435 440 445 Pro Glu His Thr Asn Leu Ser Leu Arg Ser Ser Ser TrpArg Lys His 450 455 460 Cys Gly Ser His Ser His Pro His Ser Thr Gln AlaPro Gly Glu Ala 465 470 475 480 Lys Glu Pro Leu Leu Gln Asp Thr Asn Val485 490 7 490 PRT MUS MUSCULUS 7 Met Thr Gln Gly Lys Lys Lys Lys Arg AlaAla Asn Arg Ser Ile Met 1 5 10 15 Leu Ala Lys Lys Ile Ile Ile Lys AspGly Gly Thr Pro Gln Gly Ile 20 25 30 Gly Ser Pro Ser Val Tyr His Ala ValIle Val Ile Phe Leu Glu Phe 35 40 45 Phe Ala Trp Gly Leu Leu Thr Ala ProThr Leu Val Val Leu His Glu 50 55 60 Thr Phe Pro Lys His Thr Phe Leu MetAsn Gly Leu Ile Gln Gly Val 65 70 75 80 Lys Gly Leu Leu Ser Phe Leu SerAla Pro Leu Ile Gly Ala Leu Ser 85 90 95 Asp Val Trp Gly Arg Lys Ser PheLeu Leu Leu Thr Val Phe Phe Thr 100 105 110 Cys Ala Pro Ile Pro Leu MetLys Ile Ser Pro Trp Trp Tyr Phe Ala 115 120 125 Val Ile Ser Val Ser GlyVal Phe Ala Val Thr Phe Ser Val Val Phe 130 135 140 Ala Tyr Val Ala AspIle Thr Gln Glu His Glu Arg Ser Met Ala Tyr 145 150 155 160 Gly Leu ValSer Ala Thr Phe Ala Ala Ser Leu Val Thr Ser Pro Ala 165 170 175 Ile GlyAla Tyr Leu Gly Gln Met Tyr Gly Asp Ser Leu Val Val Val 180 185 190 LeuAla Thr Ala Ile Ala Leu Leu Asp Ile Cys Phe Ile Leu Val Ala 195 200 205Val Pro Glu Ser Leu Pro Glu Lys Met Arg Pro Ala Ser Trp Gly Ala 210 215220 Pro Ile Ser Trp Glu Gln Ala Asp Pro Phe Ala Ser Leu Lys Lys Val 225230 235 240 Gly Gln Asp Ser Ile Val Leu Leu Ile Cys Ile Thr Val Phe LeuSer 245 250 255 Tyr Leu Pro Glu Ala Gly Gln Tyr Ser Ser Phe Phe Leu TyrLeu Lys 260 265 270 Gln Ile Met Lys Phe Ser Pro Glu Ser Val Ala Ala PheIle Ala Val 275 280 285 Leu Gly Ile Leu Ser Ile Ile Ala Gln Thr Ile ValLeu Ser Leu Leu 290 295 300 Met Arg Ser Ile Gly Asn Lys Asn Thr Ile LeuLeu Gly Leu Gly Phe 305 310 315 320 Gln Ile Leu Gln Leu Ala Trp Tyr GlyPhe Gly Ser Glu Pro Trp Met 325 330 335 Met Trp Ala Ala Gly Ala Val AlaAla Met Ser Ser Ile Thr Phe Pro 340 345 350 Ala Val Ser Ala Leu Val SerArg Thr Ala Asp Ala Asp Gln Gln Gly 355 360 365 Val Val Gln Gly Met IleThr Gly Ile Arg Gly Leu Cys Asn Gly Leu 370 375 380 Gly Pro Ala Leu TyrGly Phe Ile Phe Tyr Ile Phe His Val Glu Leu 385 390 395 400 Lys Glu LeuPro Ile Thr Gly Thr Asp Leu Gly Thr Asn Thr Ser Pro 405 410 415 Gln HisHis Phe Glu Gln Asn Ser Ile Ile Pro Gly Pro Pro Phe Leu 420 425 430 PheGly Ala Cys Ser Val Leu Leu Ala Leu Leu Val Ala Leu Phe Ile 435 440 445Pro Glu His Thr Asn Leu Ser Leu Arg Ser Ser Ser Trp Arg Lys His 450 455460 Cys Gly Ser His Ser His Pro His Ser Thr Gln Ala Pro Gly Glu Ala 465470 475 480 Lys Glu Pro Leu Leu Gln Asp Thr Asn Val 485 490 8 426 DNAHomo sapiens 8 agtcatactg tattttttac ttgtattttt gttgttttgt gggatttaaaaaatattttt 60 attctgagga tagttgaatc cacaggatac tgagggccag ctgtattcacaacccaaatc 120 acatabaaag cgacaagttc atacacaata ggcctattag aacaggactgttctctcttg 180 tttatcattg cagcctttct agcacaaagc ctgggacatt ctggacatttagtatgtgtt 240 aaatttctct tactacatta tttccaacag tatttactgc aatctgcaattaccttcctt 300 ttgttttgta actgtgtccc ccactagaat gtaagctctg tgcagatagtgtctcattta 360 ttgatgtatc cctggcatct aataaaacac tgacaacaca agcacccagtaaatattttt 420 tgaatg 426 9 413 DNA Homo sapiens 9 agtcatactg tattttttacttgtattttt gttgttttgt gggatttaaa aaatattttt 60 attctgagga tagttgaatccacaggatac tgagggccag ctgtattcac aavccaaatc 120 acatacaaag cgacaagttcatacacaata ggcctattag aacaggactg ttctctcttg 180 tttatcattg cagcctttctagcacaaagc ctgggacatt ctggacattt agtatgtgtt 240 aaatttctct tactacattatttccaacag tatttactgc aatctgcaat taccttcctt 300 ttgttttgta actgtgtcccccactagaat gtaagctctg tgcagatagt gtctcattta 360 ttgatgtatc cctggcatctaataaaacac tgacaacaca agcacccagt aaa 413 10 601 DNA Homo sapiens 10caggagtggc taagctaggt ggtcctacct ctgggtctct catgaagttg tagtcagcca 60aaggcttgac caaggttgga ggatctactt ccaaagtgac tcactccgtg gcatttggta 120ggaggctaca aacagttcct ggacaactgg atctctccat aggctgcttg agtgtcctga 180aaacacggaa gcaggcttcc ccaggctcca agccccaaaa tgaatgaaaa agagacccgc 240aaaggaagat gcagtgcctt ttatgaccta gcctctgaag tcaatactgt cacttctgtt 300ytgatctatc aagagtcact aagcctagtc tacactcaag gggaggggaa ttagagtcca 360cctcttccag ggaggaatat cattgaatct gtgaacatat cttagaacta ccatacctag 420tttcagtact tttaaacatt cgccattttg ctttgtccct ctcttttccc cacctacata 480tacatacaca tacatgttac tccctaacca tctgagagta gggagcatgc ggtgtatccc 540tatccctcgt gtttttctct taaggaaaag gatattctat tatacaacac ggtagttatc 600 a601 11 601 DNA Homo sapiens 11 tcttaagtta cccattactt acggaaaatgattttttact gttcccttcg gttcctgtct 60 tggttagaac acagctggag attgtgttaatagcttagga cgtctgtttc cgtgagcagg 120 taacaacttt ttgaaacaaa ttccctcatctgctgaagaa gggggacaaa aacggcccct 180 atcgcccaga accgttgcga ggatttagctagctggtgac gccggagcac gaagttgtac 240 aggtagccag cagcacccac gcgagcccgcggttaccctg gccgcgcggc tactgtagag 300 ygggctggcg gcgagcgggc ggggcggtatcacgcgggag gggcggggcc cgctcgtcgg 360 ctgatcgcac gattgtgacg cgccgccggaggcaggccgg gccctcaaga tggcggcggg 420 cgcccagagc ggctcggccc ggcagtagtggtgggacggc actagctgct ggggcctgcc 480 gccccgggag tggctgcagc agcgccaggaatcgaggatg gtaaaatgac ccaggggaag 540 aagaagaaac gggccgcgaa ccgcagtatcatgctggcca agaagatcat cattaaggac 600 g 601 12 601 DNA Homo sapiens 12ttttattacc tttctttggg gttttgtttg atttgcttta cagcagatgc tttctttcca 60aatcctgtga gttttggaaa agatcgtttt taaactttct tgtcctatta ttaaggttgt 120aattaattct tagcctgctt tgggacacaa aataaaatgt ttgcaccagc aataggtttc 180acatagaaca aatgaagact tttcttgagg gctgtgaaca tgggggctat tatcatttct 240catctttata cacttaatat ttcattctct attctaagag cactgggcac tcctttagaa 300waggggcttt gttttgtatg tttggatccc acagggccta gtatgtgaat tttaaagtga 360taaaaacact tctattttgt actagcacat tcctagatga atttttattg taattttgtt 420tattcttata cgtaatcaga ggatatattt caataaatat caggggaata ttttgcatta 480tttgtatttt aatccatccc agctttaaat ttaaaaagta taactattgc agtcatagaa 540atgattgtaa aatggtagtt gcttatctac ctctctactt acaatagttc agactactat 600 t601 13 601 DNA Homo sapiens 13 taatccatcc cagctttaaa tttaaaaagtataactattg cagtcataga aatgattgta 60 aaatggtagt tgcttatcta cctctctacttacaatagtt cagactacta ttatgaactt 120 tttttgtttg tttgtttgag atggagtctcactctgttgc ccaggctgga ggagtgcagt 180 ggcaggatct cggctcactg taaccaccgcctcctgggtt caagtgattc tcctgcctca 240 gcctcccgag tagctgggac tacaggcacgtgccaccatg cctggctaat tttttatatt 300 ktcagtagag acaaagtttc accatattggtcaggctggt cttgaactcc tgacctcatg 360 attcacccac cttggcctcc caaagtgcagggattacagg tgtgagccac cgtgcccaga 420 ctgaacattt tttaagaaag gggaaaaaattgccatttga tactctgttg ttgtgtgttt 480 tttaattcat cgtatcatag aatatttcagtgctattgct gttgacctca gagtttcaga 540 gtttttataa agttccgcca atgggtagattcattcagtg agatgtctga ggctctatgg 600 t 601 14 601 DNA Homo sapiens 14caagtgattc tcctgcctca gcctcccgag tagctgggac tacaggcacg tgccaccatg 60cctggctaat tttttatatt ttcagtagag acaaagtttc accatattgg tcaggctggt 120cttgaactcc tgacctcatg attcacccac cttggcctcc caaagtgcag ggattacagg 180tgtgagccac cgtgcccaga ctgaacattt tttaagaaag gggaaaaaat tgccatttga 240tactctgttg ttgtgtgttt tttaattcat cgtatcatag aatatttcag tgctattgct 300bttgacctca gagtttcaga gtttttataa agttccgcca atgggtagat tcattcagtg 360agatgtctga ggctctatgg tcggtacatg acagtcgtga acagtatttc acatacctgg 420tcaatggtac tgatttgatc ccccttctga tttcttcttt tcaacaatgt taataaaatt 480ctttcccgtt gtcctgctaa tgacatatat gtaagcctat ttggccagtt taaatattta 540taaacaaaac tagtaagagt tgttaatgat ttttctgaaa attagagcag attagagcag 600 a601 15 601 DNA Homo sapiens 15 cttatctttt aaaatgctca tcttaaaatatgatctttat tgttttggcc atacaattgt 60 ggaactacat ctctgacagt ggaaaatgtatagttctttc agaagtttgt ggtaaaatga 120 ctttaaagat ttgatagaaa gtaaggcatatctgaattgc atggtcggaa gtacctgaaa 180 aaagtaaaat tgatatatca tttgaaaatgaaatgcatat ccctggataa gcagagcacc 240 agattttttt tttcttggca tccctgattttaattaaata ggagtcagca accgtttcaa 300 ragcaggacc caagctctga ccctttgcactcttcacctg caaggatggc tgaagtagtg 360 gcaggaaagc tctctgggat gtagggcctttgtagaccca gagagctgtt aaataacctt 420 tggttgctag catgcaagca ataagaagggcctgtggtgc ttttcttttt ctttcttttt 480 ttttttcttt tgagacagag ttttgctcttgttgctcagg ctggggtgca atggcgtgat 540 cttggctcac agcaacctct gcctccctggttcaaggaat tctcctacct tagcctcctg 600 a 601 16 601 DNA Homo sapiensvariation (301)...(301) A may be either present or absent. 16 taggacttctaaatttttta attactatgg gtacaaagta gatacagata tttatcaggt 60 acatctgatattttgataca agcatatgtt gatacaggta tacagtgtat aataaatcag 120 ggatactggggtatccatta cctcaaactt ttatcatttc tttgtgttag gaacatgcca 180 attccacttttattttattt tattttttat tttttgagac agagtctcgc tctgtcgccc 240 aggcgacatacatagtacag tagtgtactc cagcctgggt gacggggaga ctctgtctca 300 aaataaataaataaataaat aaataaatct gttcagacta atgtcctaga gtgtattccc 360 aatgttttcttctagtcgtt tgtggtttca ggttttagat ttaagtcttt aatccatttt 420 gatttgattgttgtacatgg caagaggtag gggtataatt ttattcttct gtatatggat 480 atccacttttcctagcacca tttaggagac tatccttttc ccaatgtata cttcggtgcc 540 gttgtcaaaaatgagttgac tgtaaatgca tggatttatt tctgggttct ctattgtgct 600 c 601 17 601DNA Homo sapiens variation (301)...(301) T may be either present orabsent 17 tgggtacaaa gtagatacag atatttatca ggtacatctg atattttgatacaagcatat 60 gttgatacag gtatacagtg tataataaat cagggatact ggggtatccattacctcaaa 120 cttttatcat ttctttgtgt taggaacatg ccaattccac ttttattttattttattttt 180 tattttttga gacagagtct cgctctgtcg cccaggcgac atacatagtacagtagtgta 240 ctccagcctg ggtgacgggg agactctgtc tcaaaataaa taaataaataaataaataaa 300 tctgttcaga ctaatgtcct agagtgtatt cccaatgttt tcttctagtcgtttgtggtt 360 tcaggtttta gatttaagtc tttaatccat tttgatttga ttgttgtacatggcaagagg 420 taggggtata attttattct tctgtatatg gatatccact tttcctagcaccatttagga 480 gactatcctt ttcccaatgt atacttcggt gccgttgtca aaaatgagttgactgtaaat 540 gcatggattt atttctgggt tctctattgt gctctattgt ctatgtatctgtttttatac 600 c 601 18 601 DNA Homo sapiens 18 tttgttttta atttttttgagacggagttt tgctcttgtt gcccaggctg gaatgcaatg 60 gcgcaatctt ggctcaccgcaacttccgcc tcccgcgttc aagcgattct cctgcctcag 120 cttcctgagt agctgggattacaggcatgc gccaccacgc ctggctaatt ttgtattttt 180 agtggagacg gggtttcttcatgttggtca ggctggtctt gaactcctga cctcaggtga 240 tccacccgct ttggcctcccaaagtgctgg aattacaggt gagagccact gcgcccggcc 300 batgaatgat cttttttaagacctccttcc tgaaggaggt ttgctagtat tttgttgagg 360 atttttgcat caatgttcatcagagatatt gtcccatagt ttattttgtt tttctccatg 420 ctagttttag gtaatttttctcttaaataa acaaagcatt ttcctcctaa agtgcaagca 480 tgcttattag aaaagatatggaaaattcag aatagcatag taaacaatgt gatatcactt 540 aaaatcatta cctaatataaattttattta cattgaggtc agtatttatt gtttttcaga 600 g 601 19 601 DNA Homosapiens 19 ccgcaacttc cgcctcccgc gttcaagcga ttctcctgcc tcagcttcctgagtagctgg 60 gattacaggc atgcgccacc acgcctggct aattttgtat ttttagtggagacggggttt 120 cttcatgttg gtcaggctgg tcttgaactc ctgacctcag gtgatccacccgctttggcc 180 tcccaaagtg ctggaattac aggtgagagc cactgcgccc ggccgatgaatgatcttttt 240 taagacctcc ttcctgaagg aggtttgcta gtattttgtt gaggatttttgcatcaatgt 300 ycatcagaga tattgtccca tagtttattt tgtttttctc catgctagttttaggtaatt 360 tttctcttaa ataaacaaag cattttcctc ctaaagtgca agcatgcttattagaaaaga 420 tatggaaaat tcagaatagc atagtaaaca atgtgatatc acttaaaatcattacctaat 480 ataaatttta tttacattga ggtcagtatt tattgttttt cagagttgaaattaccctac 540 ctatacatgt tatatcctac tttgattttt aaaaaaatta gcatgctttaagccctgaga 600 a 601 20 601 DNA Homo sapiens 20 gttcttgatt ctcccttccgctatttatca agctcctccc aatttcaaat cctgaatcct 60 taatccgttc cctcccctccaacattcata ctgtgccact gttttatgcc ctcatttctt 120 gtttgagctg attcagatagcttcctttta gatgcgcttt gcttctccat tttatccttt 180 aggaaatcac cagagtgataatactgcagt gagtcttaag acatctctgg cagcggtata 240 aacttaattt tgtattttctttctcatgta tatcaaattc caaatctctt acatactttc 300 sctggggatt gttctgcttttgagccatgt tgatatcgtg tttatatttt tgccacttgc 360 ttcatttatg gtttttttttttttttttgg ttacatcttt gccagaataa tcttaaaact 420 ttcatctgat tgtgtcagtcttaatatctt ttagtggctc cccatggcct tcagaattaa 480 atatagactc cttagcatggaagctggtct ttgagtacct gtagcttgtc tttcaataca 540 cccaacgtgc agcccatgcactggttgtac tgaactcgat atatgagacc cataatgccg 600 c 601 21 601 DNA Homosapiens 21 acaggctgag ccatcttttc caccctatac ctccgcctgt ctaactctgttgtgtccttt 60 cagccttcct cctggaagtc tgatatttcc cacctcccaa gctcccttggactctgtatg 120 ttccaactgc atactgtgct tatgctaatg aatttcgttg ttgccttgtctgtccctctg 180 actttgaaga cagaggcagt gagtacagat gtttgacaca gtgcccagtacatatatgat 240 cttaatattt gttgactatt aacatcgttg ttattgttaa taattatagaatgtactgtt 300 wacttttttt aactttttaa aaaatcttgt tttttatagc ctcaaggaataggttctcct 360 agtgtctatc atgcagttat cgtcatcttt ttggagtttt ttgcttggggactattgaca 420 gcacccacct tggtggtaag taatctttta aattatttaa cactgactccaaaatctctt 480 cttcttcagt tttggaggaa aatgtgggcc ttttcccttt gcacggttaattctcccacc 540 agtattgttc agtattcacc agtattttac tggttgtctt ttccaactgttaactctccc 600 t 601 22 601 DNA Homo sapiens 22 gaggaaaatg tgggccttttccctttgcac ggttaattct cccaccagta ttgttcagta 60 ttcaccagta ttttactggttgtcttttcc aactgttaac tctcccttac ctttttttgg 120 gaggggggtg gcgtggaggtgtttgaattt ggacttgtca ctgggcatgt tcaagcagag 180 gctctgtaac tactctgagtaaaatggaag agattcttaa accgacaggt ttagaaaaga 240 tgatgtctgt gacctgcatgactcggcata attactttga ggttcattta tgcagctgta 300 vtttccaaaa acaggtttctgttcatttgg gctaagtacc tagaagggct attctttaat 360 agatctaagc tgattttacccaaattctcc caggtttgaa actttagaaa agacctccct 420 gcccgaccaa acaactcagaagatagccag ttttcttata ttggtgtaga taaggggaat 480 ggaaggaggg aaggactatctatggtaaat atctatacca tcttgaaagg agtaattatg 540 ataaatgtac agtttaccaaatcctagagg aatagagttt taaagtaata tactatgttt 600 t 601 23 601 DNA HomoSapiens 23 aaaaaaaaac agccgggcgc ggtggctcac acctgtaatc ccagtactttgggaggccaa 60 ggcgggtgga tcacgaggtc aagagattga gaccatcctg accaacatggtgaaaccctg 120 tctctactaa aaatacaaaa attagctgtg cgtggtggta cgcacctgtaatcccagcta 180 cttgggaggc tgaggcagga gaatctcttg aacccgggaa gtggaggttgcagtgagccg 240 agactgcacc actaccctcc agcctggata cagggtgaga ctctgtctcaaaaataaaaa 300 rtcattttga atatatagag catgttcatg agtattgcta taaaaaaatatcagagggtt 360 tttttttttt ttttagttta ctgatttcag atagaaatct ttaaaaaattaatttacaca 420 tttcctggct tcataatcca agtacaacga tttggaactt cctcagatgatgcaagttga 480 ttatgacatt cataacttca ttgaattgta ataacctgtt tttgtcaagggttactgaag 540 tgctgtaata actttttggg ctcatgactt tacattagct ttcctaatgcgccagcgtgc 600 t 601 24 601 DNA Homo sapiens 24 cttgactcct ccagtgctcagtcttttggg gaatgcaggt agtaacttgt ttgtacccat 60 gttttagata gttgaggttgtcaggcagcc caaccactag ctaagtaggg tgatcaaaat 120 gtggatgagc tgttagcaagctatgaaaaa aagcattttg tgatgtttcc ataatttgtt 180 atcagtattt caagtgtgtatagctatttt taaaatttgc ttcttgttta aattttttta 240 ggtatgttat ctttcgtgttattttggtac atttttttcc tagttggaca aagggaggct 300 htctttttta agaacaaggaaggagtcccc ttaattagaa aggcttgttt attcattttt 360 catagactaa tgtgcttaatatattccttt tttttttttt tttttttttt gagacggagt 420 ctcgctctgt ctgtccccaggctggagtgc agaggcacga tcttggctca ctgcatcccc 480 cacctcccag gttcaagtgattttcctgcc tcagcctccc aagtagctgg gactacaggc 540 acatgccacc atgcccagctaatttttgta cttttagtag agatggggtt tcaccatgtt 600 g 601 25 601 DNA Homosapiens variation (301)...(301) T may be either present or absent 25accactagct aagtagggtg atcaaaatgt ggatgagctg ttagcaagct atgaaaaaaa 60gcattttgtg atgtttccat aatttgttat cagtatttca agtgtgtata gctattttta 120aaatttgctt cttgtttaaa tttttttagg tatgttatct ttcgtgttat tttggtacat 180ttttttccta gttggacaaa gggaggctat cttttttaag aacaaggaag gagtcccctt 240aattagaaag gcttgtttat tcatttttca tagactaatg tgcttaatat attccttttt 300tttttttttt ttttttttga gacggagtct cgctctgtct gtccccaggc tggagtgcag 360aggcacgatc ttggctcact gcatccccca cctcccaggt tcaagtgatt ttcctgcctc 420agcctcccaa gtagctggga ctacaggcac atgccaccat gcccagctaa tttttgtact 480tttagtagag atggggtttc accatgttga ccagaatggt ctcgatctct taacctcgtg 540atccgcccgc cttggcctcc caaagtgctg ggattacagg tgtgagccac tgtgcctggc 600 c601 26 601 DNA Homo Sapiens misc_feature (1)...(601) n = A,T,C or G 26ttcaaagata ttctttgaag tattttttta atcagataac cagttttaga catattaatt 60ttgaatgtct ggtttgggat ttatgatagc cttaatttct taatttttaa aactaatgtg 120acattttaag accaaaaaaa ctgtgtgttg caattatctt tcacttttaa gccctcatag 180aacagtcaaa aaacaaaagc tgtgttttgt ggaagatctg cccaggggaa gatggtgagc 240ctctaccaac aaggggattt agctaaaaag aaggattttg tactgacaaa tatttttaaa 300nattgaggtc taacactttt gagaggttat gaatatatgg ttggtcatag tagatagttc 360agtcagaatc agtgattatt gcttgattat gtaacatatt agctaagtga tgagaataac 420agtaggtata aggatctgta atgccaagga gtggaattta ccggtttttt tttttctttc 480cttttttttt tttttcattg agacggagtc ttaatctggc atccaggttg gagtgcagtg 540gcgtgatctc ggctcactgc aacctccacc gccaaggttc aagagattct cctgcctcag 600 c601 27 601 DNA Homo sapiens variation (301)...(301) T may be eitherpresent or absent 27 aaaagctgtg ttttgtggaa gatctgccca ggggaagatggtgagcctct accaacaagg 60 ggatttagct aaaaagaagg attttgtact gacaaatatttttaaagatt gaggtctaac 120 acttttgaga ggttatgaat atatggttgg tcatagtagatagttcagtc agaatcagtg 180 attattgctt gattatgtaa catattagct aagtgatgagaataacagta ggtataagga 240 tctgtaatgc caaggagtgg aatttaccgg tttttttttttctttccttt tttttttttt 300 tcattgagac ggagtcttaa tctggcatcc aggttggagtgcagtggcgt gatctcggct 360 cactgcaacc tccaccgcca aggttcaaga gattctcctgcctcagcctc cccagtagct 420 ggaattacag gtgcatgtca ccacgcccag ctaatttttttttttattat tttttttgag 480 acagagtttc actctgtcgt ctaggctgga attcagtggcactatctcgg ctcactgcaa 540 ccttcgcctc ccaggttcaa gcagttctct gcctcagcctcccaagtatg tgggattaca 600 g 601 28 601 DNA Homo sapiens 28 cccacctaagcctcccaaag tgctgggatt acaggcgtga gccactgttc ctggccggct 60 ttacccttttgacagaccta tggctctgga aataataggc cagtgtttga tggttcaagc 120 tcctagatacacagtccatg ttacggaaca ctcaaaatcc actagcatct cttctaccta 180 gatggtttcgtgtccttggc tacagaaaca gccccaaagc gtttaacatt ttaaggatta 240 tttactttcaacatttttaa agttaaaaaa aagttaagat ccataaaatt ttttggaaaa 300 rtgttacattttctctgttc acctctaaag accagtgcta aaggatcctg acatcaaaaa 360 tctttacaacattcgaatta cttgttatat ttgtctgtta aaattttgtt agaaattgta 420 tggccccaaaggagaaattg ctttggagaa aaaagttagg tagcagagga acagtttgga 480 agggttgggggttggccaga taaagaaagg gaagaaacat tcaaaattga aaggatgccg 540 tgtataaaatatgaatattg gaaagcatag aatatttcag aaacagtgaa gcgaacagat 600 t 601 29 601DNA Homo sapiens 29 aggaggctga ggcaggagaa ttgcttgaac ctgggaggcggaggttgcag tgagccaaga 60 ttgcaccatc gcactccagc ctgggcgaca agagcccaactccgtctcaa acaaacaaaa 120 aaaggaatag tgctgcagta aatgtaggag tacagctatctcttcaatat actgatttcc 180 tttttttgga ggggtatata cctagtagtg agattgctggatcatatggt agctccattt 240 ttaggttttt tgaggagcct tccaactgtt ttccttagtgattgtactaa tttacattcc 300 vaccaacagt gtatgagtgt tcccttttct ccacatccttgcctatcttt tggataaaag 360 ctgtttttaa ctggggtgag atgatatttc actgtagttttgatttgcat ttccccgatg 420 atcagtgatg gttgagcatt ttttcatata cctattggtcactttgagaa atgtctattc 480 agatcttttg cccgtttttt aaaaatcaga ttatgagattcttttcttac agaattgttt 540 gagcccctta tacatttttg ttattaatcc cttgtcagatggatagtttg cagatatttt 600 c 601 30 601 DNA Homo sapiens 30 gcacatttttgttaaatttc tcgctattgg caggagaaga ataactgaag aaaggggagc 60 aattctgatccttctaaagg ttcttcttgc aacatgtcag aaagtatatt tagcataatg 120 tttcttcttaaagggaagac cttccctacc ttccttatta cccacattcc cattctctgt 180 tgttattactgagcgatagc attggataat agaagcatta gtttctaagt caaacaggaa 240 ctcagttgcctcatatgtaa agtgataata ttatctaatt cacagtgttg ggattaaaca 300 rgagtacatataggctgtaa aaatggtagc tgctgtttat ttttccagtt gcctggaatt 360 gccttttcatttgatgcatt ccagcggttc tcttgctgcc cactgcaaaa aattgatacc 420 acatgatttgagaacaagcc ttggaaagga tagaataact tgttatacat tttcataggt 480 tgggattttttttctttata gaatctttct agatctactt cgtggcaatt aaaaattact 540 tattaattttcccaatctcc tatcctagat aatatatcca tctgaaagag aattataagt 600 c 601 31 601DNA Homo sapiens variation (301)...(301) A may be either present orabsent 31 agacaaatac cttgtgataa ataaggactg aatattgtgt tgggctgaattagttttaaa 60 agggactgat ttctgattca aaggacgtta tagtgaagaa tcataagatttttggggagg 120 aaacacctat agagagaaag ttagaaaaag aactaataat ttctggcctgttcagtggct 180 cacacctgta atctcagcac tttgggaggt tgaggcaggc ggatcacttgagatcaggag 240 ttcacgacca gcctggccaa catggtgaaa ccttgtctct atttaaaaaaaaaaaaaaaa 300 aaaaagtgaa aagaaaaaga actaatgatt tcagttgtaa acttggaacattaaatgata 360 caaggctgat gatagccagg atatttaaaa aatagtctaa ttaagctatagtttacatac 420 cataaaattt atccttttta tgagtatagt tcagtgaatt ttagtaaatttatactgtta 480 tgcaaacacc accataaccc aatttggggt tggtcggttg gttggttggttcgttggttt 540 ggtttttttg acgtaattta ttttcccata gccaaagttt tgaaattaacaattttcaat 600 c 601 32 601 DNA Homo sapiens variation (301)...(301) Amay be either present or absent 32 gttgtaaact tggaacatta aatgatacaaggctgatgat agccaggata tttaaaaaat 60 agtctaatta agctatagtt tacataccataaaatttatc ctttttatga gtatagttca 120 gtgaatttta gtaaatttat actgttatgcaaacaccacc ataacccaat ttggggttgg 180 tcggttggtt ggttggttcg ttggtttggtttttttgacg taatttattt tcccatagcc 240 aaagttttga aattaacaat tttcaatctggaggttctgt gtattaagcc atgttctggc 300 aaaaaacaaa acaaaacaaa acaaaacaaaacaaaacaaa aaacactgaa atcttctaga 360 aataatatgg atgcagaaaa aaggtggggaagtggccagg cacagtggca tgtgcctgta 420 ataccaccag tttgggaggc caaggcagggggattgcttg aggccaggag tttgaggctg 480 cagctatgat catgccacta cactccagtctagggtacag agtgagaccc tgtctcttaa 540 aaaaaaaagt tggaggggcc aggtgcagtggcttataatc ccagcacttt gggaggctga 600 g 601 33 601 DNA Homo Sapiensvariation (301)...(301) C may be either present or absent 33 aacttggaacattaaatgat acaaggctga tgatagccag gatatttaaa aaatagtcta 60 attaagctatagtttacata ccataaaatt tatccttttt atgagtatag ttcagtgaat 120 tttagtaaatttatactgtt atgcaaacac caccataacc caatttgggg ttggtcggtt 180 ggttggttggttcgttggtt tggttttttt gacgtaattt attttcccat agccaaagtt 240 ttgaaattaacaattttcaa tctggaggtt ctgtgtatta agccatgttc tggcaaaaaa 300 caaaacaaaacaaaacaaaa caaaacaaaa caaaaaacac tgaaatcttc tagaaataat 360 atggatgcagaaaaaaggtg gggaagtggc caggcacagt ggcatgtgcc tgtaatacca 420 ccagtttgggaggccaaggc agggggattg cttgaggcca ggagtttgag gctgcagcta 480 tgatcatgccactacactcc agtctagggt acagagtgag accctgtctc ttaaaaaaaa 540 aagttggaggggccaggtgc agtggcttat aatcccagca ctttgggagg ctgaggcagg 600 a 601 34 561DNA Homo Sapiens 34 agcagaaaga aatggctacc aagtggagag aactgaggagaagggaaatg acatgaaaca 60 actgtactga cttgctcact gtgtcacaaa tgtgatctctgtaaatgccc tcaaatgtct 120 tcagtgaccc tcatagtgag aaccattttc cctttccccacacttgtgcc agagccctgc 180 tgagatctgg gtccctctga aaccacacct agggctgcaataacaaaata accactacat 240 ttgaaaatat atatttatat gtatgtgtgt gtgtgtatgtrtgtgtgtgt atatatatat 300 agtttgtttt ttgttgagac ggagtctcgc tctgtcacccaggctggagt gcagaggtgt 360 gatcttggct tactgcaacc tccgcctcct gggttcaaacgattctgctg cctcagcctc 420 cccagtagct atgcccacca ccatgcccag ctaatttttgtatttttagt agagacgggg 480 tttcaccata ttggccagtc ttgtcttgaa ctcctgacctttggtccgcc tgcctcggcc 540 tcccaaagtg ttgggattat a 561 35 385 DNA HomoSapiens 35 agcagaaaga aatggctacc aagtggagag aactgaggag aagggaaatgacatgaaaca 60 actgtactga cttgctcact gtgtcacaaa tgtgatctct gtaaatgccctcaaatgtct 120 tcagtgaccc tcatagtgag aaccattttc cctttcccca cacttgtgccagagccctgc 180 tgagatctgg gtscctctga aaccacacct agggctgcaa taacaaaataaccactacat 240 ttgaaaatat atatttatat gtatgtgtgt gtgtgtatgt atgtgtgtgtatatatatat 300 agtttgtttt ttgttgagac ggagtctcgc tctgtcaccc aggctggagtgcagaggtgt 360 gatcttggct tactgcaacc tccgc 385 36 361 DNA Homo Sapiens36 agcagaaaga aatggctacc aagtggagag aactgaggag aagggaaatg acatgaaaca 60actgtactga cttgctcact gtgtcacaaa tgtgatctct gtaaatgccc tcaaatgtct 120tcagtgaccc tcatagtgag aaccattttc cctttcccca cacttgtgcc agagccctgc 180wgagatctgg gtccctctga aaccacacct agggctgcaa taacaaaata accactacat 240ttgaaaatat atatttatat gtatgtgtgt gtgtgtatgt atgtgtgtgt atatatatat 300agtttgtttt ttgttgagac ggagtctcgc tctgtcaccc aggctggagt gcagaggtgt 360 g361 37 601 DNA Homo Sapiens 37 agtagagacg gggtttcacc atattggccagtcttgtctt gaactcctga cctttggtcc 60 gcctgcctcg gcctcccaaa gtgttgggattataggcgtg agccatggcg cctggccccc 120 atgtgaatat attaaatacc atttaaaaaaccaccacaac ccagttatag aacatttcca 180 tcagcccaaa atgttccctc agccctgtttgccatctgtc cccatgctcc acctgtgacc 240 ccaagcaacc aacaatttag cttctgtcaccatggttttg ccttttctag aaacttcata 300 kaaattaaat aatacaaaac atcttttgtgtctaacttct ttcacttggc ataatctttt 360 gagattgatc catgttgata ctatagatcaataggttcta tttttgtctc ttttcctttt 420 tttttttttt gagacagggt cctgctctatcccccaggct ggagtgcagt ggcatgatca 480 tggctcactg aagccttggc ttcctgggctcaagcgatcc ttctgcctca gcctccaaag 540 cagttgggac cacaggcatg atccaccatgcccagctaat ttttttcttt ttgagacagg 600 g 601 38 601 DNA Homo Sapiens 38gcccttaacc aagcttgtcc aacccatggt ccacaggctg cacacatggc ccaacagaaa 60ttcataaagt ttcttaaaac attatgcagt ttttttttct ttaagctcat cagctattgt 120tagtgtattt tatgtgtggc ccaagaccgt tcttccagcg tggcccaggg aagccaaaag 180attagacacc cctcccctaa ggaccagcat gactggcagt caaggagggg tgtttgtaca 240gtgcccaggc tctcaaccct tcctcaacta aaagagttaa aaaatttaaa taggccgggc 300rtggtggctc acgcctgtaa tcccagcact ttgggaggcc gaggcgggcg gatcacgagg 360tcaggagatc gagaccatct tggctaacac gggggaaacc ccatctctac taaaaataca 420aaaaattagc cgggcgaggt ggcgggcgcc tgtagtccca gctattcggg aggctgaggc 480aggagaatgg cgtaaacccc gggggcggag cctgcagtga gccgagatcg cgccactgca 540ctccagcctg ggcgacagag cgagactccg tctcaaaaaa aaaaaaaaaa aaaaaaaaaa 600 t601 39 601 DNA Homo Sapiens 39 ctttaagctc atcagctatt gttagtgtattttatgtgtg gcccaagacc gttcttccag 60 cgtggcccag ggaagccaaa agattagacacccctcccct aaggaccagc atgactggca 120 gtcaaggagg ggtgtttgta cagtgcccaggctctcaacc cttcctcaac taaaagagtt 180 aaaaaattta aataggccgg gcatggtggctcacgcctgt aatcccagca ctttgggagg 240 ccgaggcggg cggatcacga ggtcaggagatcgagaccat cttggctaac acgggggaaa 300 vcccatctct actaaaaata caaaaaattagccgggcgag gtggcgggcg cctgtagtcc 360 cagctattcg ggaggctgag gcaggagaatggcgtaaacc ccgggggcgg agcctgcagt 420 gagccgagat cgcgccactg cactccagcctgggcgacag agcgagactc cgtctcaaaa 480 aaaaaaaaaa aaaaaaaaaa aatttaaatagaggcagggt cttgctgtgt tgcccaggct 540 ggtcacaaac ttctggcttc acgcagtcctcccaccttgg cctccccaag tgctgagatt 600 a 601 40 601 DNA Homo Sapiens 40ccaggcttgg tgtctcatac ctgtactccc agcccttcgg gaggctgagg tgggaggatc 60gcttgagctc aggagttcga gactagccta ggcaacatag cgtgacttcc acctctataa 120aaaataaaca aaattagctg ggcgtggtgg tgtgtgcctg tagccccatc aggagatctt 180caggcaggaa gatctacttg agcctgagag gtcaagacta cagtgagccg tgatggcacc 240actgcactcc agcctgggcg acagagcaag acccagttcc cccactctcg cccccacaag 300raaaaaagat aaatggcaca ggtaggaaga gaaaagggag ggtgtgcaac agaaggcctg 360acataaatca agattatgaa aggagttatg tggtgttgag gaaaaaagta gcctgactaa 420tctctgtcta tccttaattt attgcaggtt tcagcaacat ttgctgcaag tttagtcacc 480agtcctgcaa ttggagctta tcttggacga gtatatgggg acagcttggt ggtggtctta 540gctacagcaa tagctttgct agatatttgt tttatccttg ttgctgtgcc agagtcgttg 600 c601 41 601 DNA Homo Sapiens variation (301)...(301) R may be eitherpresent or absent 41 tactatttct agcttcaggt ttgtcctaaa aatcatcagtctggaaaaac aatgcattta 60 aatattcatt cctagccatg agaaaagtgc tttttaactttggaggaaaa tatactgtag 120 cctttatata aaaatggctt taaaaaaagt ttttgaggccaggtgcggtg gttcatgtct 180 gtattcccag cactttgggt ggccaaggtc agggaccgcttgagcccagg agttcaagac 240 cagctcaagc aacttggcaa aaccccatct ctaccaaaaaaaaaaaaaaa aaaaaaaaaa 300 rgaaagaaag cccggtgtgg tggtgtgtgc ctgtagtcccagctactcag gaagctgaga 360 tgggaggatt gcttgatcct gggaggtcga gggtgcagtgagccacagtt gtcccatggt 420 actccagtat gggcaacaga atgagaccct gtctcaaaaaaaaagtgtaa ggaaaataca 480 cagttagtat gtgtagaact tgatgaatta tcaaagattaacccaacctt gcaatagatg 540 tgatcaacct gggcatgagt atctttttca tacattgccaacattagatt tgctaacatt 600 t 601 42 601 DNA Homo Sapiens 42 tcagccccattacttcctgg gcaccggtac gtataagagt tcctaacact tgcactaagt 60 aagtgtttacatgagaacat caatatagtt ctacacattt cttttttctc agtgttttcc 120 tatccagccctctctgtggg ggtgactccc acacttactc ttccagtcca gtccctgagc 180 tcctatatgacacattggct gggtatctca gccttaacat ggccaaaatt aaaatctggg 240 ttccatcccttgcccgccac ccctatgctc ctcatctcat tcagtggctt caccaccgcc 300 wggttttgggggccagaagc cttagcgaca ttcatgaatc ctttctccct aaccttacat 360 tcagcccatcaaatgatact tcctatcacc tctctctcca aaatatatct tgaatcagag 420 cgtttctgatattctccatt agtaggaacc caatctgagc cgtggccata tcttccatct 480 ggtctctctgcttccatctt gcctcactat agttcattct gcacttggca gagtaatttt 540 tgtaaaatggaaatttaatc gcatcttacc tataactcac tttcctttgc aaccagaata 600 a 601 43 601DNA Homo Sapiens 43 attagttaga acttttgacc cttccatttt caactactgatttaagtggt cctcagtaga 60 aatgtactga ataggaagtt ttatctttca gttttctaactctcagtctg gatctatgtc 120 agcagaggga cttttcatct gcttatgtga cctggactagtgatctcaga catattcagg 180 gcaattattg ctgaaaatca gccaaattgt agaaaagtgccaatagtcct tttatagtgt 240 agattgaaag aagtcacttt ttaaaacttt attctgataaatcttttttt ttttttttca 300 raccatagtc ttgagtttac ttatgaggtc aattggaaataagaacacca ttttactggg 360 tctaggattt caaatattac agttggcatg gtatggctttggttcagaac cttggtaagt 420 ataaatattt taatgttaat atttttaatt ttggtgttagcccttgtgtt tttatttgct 480 tctcaactga ggggtagact gtaatctgtc tcatactatgctttttatct ttcaaaatgt 540 gtctaatata agtctgccac ttgtatattt atatgttctcctagaatggc ttgaggatta 600 a 601 44 601 DNA Homo Sapiens 44 taagtggtcctcagtagaaa tgtactgaat aggaagtttt atctttcagt tttctaactc 60 tcagtctggatctatgtcag cagagggact tttcatctgc ttatgtgacc tggactagtg 120 atctcagacatattcagggc aattattgct gaaaatcagc caaattgtag aaaagtgcca 180 atagtccttttatagtgtag attgaaagaa gtcacttttt aaaactttat tctgataaat 240 ctttttttttttttttcaga ccatagtctt gagtttactt atgaggtcaa ttggaaataa 300 raacaccattttactgggtc taggatttca aatattacag ttggcatggt atggctttgg 360 ttcagaaccttggtaagtat aaatatttta atgttaatat ttttaatttt ggtgttagcc 420 cttgtgtttttatttgcttc tcaactgagg ggtagactgt aatctgtctc atactatgct 480 ttttatctttcaaaatgtgt ctaatataag tctgccactt gtatatttat atgttctcct 540 agaatggcttgaggattaaa aaggtgacct tttatagcta aatgacaggc tgaatttttg 600 a 601 45 601DNA Homo Sapiens 45 tctggatcta tgtcagcaga gggacttttc atctgcttatgtgacctgga ctagtgatct 60 cagacatatt cagggcaatt attgctgaaa atcagccaaattgtagaaaa gtgccaatag 120 tccttttata gtgtagattg aaagaagtca ctttttaaaactttattctg ataaatcttt 180 tttttttttt ttcagaccat agtcttgagt ttacttatgaggtcaattgg aaataagaac 240 accattttac tgggtctagg atttcaaata ttacagttggcatggtatgg ctttggttca 300 raaccttggt aagtataaat attttaatgt taatatttttaattttggtg ttagcccttg 360 tgtttttatt tgcttctcaa ctgaggggta gactgtaatctgtctcatac tatgcttttt 420 atctttcaaa atgtgtctaa tataagtctg ccacttgtatatttatatgt tctcctagaa 480 tggcttgagg attaaaaagg tgacctttta tagctaaatgacaggctgaa tttttgaatg 540 agattataca gcttttgaat ctttaaggag catttaatctaaatcagtcc gttactagga 600 a 601 46 601 DNA Homo Sapiens 46 tcatgtgtttggaaatattt gatgatgttt cgtcatctat attatacatt atcctcaata 60 taacctctcaattgcctgta gaaataatac ccagcacatt ttacagtttg gaacatgata 120 tccctattttacattacacc ctcacagcac tcttgtgaat taagttgctg tctttgtata 180 cagggtcagattgttaagcg acttgcccat agtcacctag taagcaagtt cagttctcct 240 tttctctacagccatttcac agtaagaatt acttaattat gtagtttgac tttcaggtac 300 rgtggagaagaatttactgt ttttgttttg ctgctctcct tataggatga tgtgggctgc 360 tggggcagtagcagccatgt ctagcatcac ctttcctgct gtcagtgcac ttgtttcacg 420 aactgctgatgctgatcaac agggtgagtt gataggaact agcgataatt atttaaaagt 480 acagaatgttctaatcctgt gttctgtctc ctatgtactg aaacataagt atatcttcag 540 ggtagagacttttaaaattg cttttgatat aaacaggaaa agcagattct agggtattta 600 t 601 47 601DNA Homo Sapiens 47 cttaggtaga tacatattcc cttttctctc acttagaatatgtggcttat ctgttctgtt 60 catagaaaat ttactgatga ggctgggcat ggtggctcacacctgtaatc ccaacacttt 120 gagaggccga ggcaggcaga ttgcttgagt tcaggagttggagaccagcc tgggcaacat 180 ggggaaaccc catcactaaa atgcaaaaat tagctgggcagggtggcgca tgcttgtagt 240 cccagttact tgggaggctg aggttggagg atcacttgagtctcagaggc ggaggttgca 300 stgagccaaa atcaggccag tgaactccaa cctgggcgacacagtgagag accttgagag 360 acctgtctca aaaaaattta ctgatgaatg ttggccacagaatattaact aagcattaag 420 tttttgctgt gttgtgctag acaccttgtg ggatatattcatagaccttt tatgaatgtt 480 gtcccagcca cttgggaagc tgaggcagga ggattacttgagctcaagag tttgaagcta 540 gcttaggcaa cacagcaaga ctcccatctc taataaaaaaaaaaaaagaa atcatatagt 600 t 601 48 601 DNA Homo sapiens 48 agcttttatgtgagattcta tttgagattg gataaaatgc ttaaaaacca gagtttaagg 60 gactgtgttcttctacatac caccttgtga aaattggctg tgcatatttt ttttccactt 120 gagaatgaaaaattttaagt acctagtttg tcaatggcat atgtaacaaa ccatttctct 180 ttactactagtctcttttga aacttttcta atatcacagt tgtgtatgtt aactaacttt 240 tcatacaaaaggcaggctta tggtaaataa cctttgttca ctgtttgcta tatttccctc 300 dtttcaactgaaaattaatg ccaaacaatg ctgatcattt tggccaatat tagcagctca 360 tcagtttcctggcttattta gcagagttct gtacttatct tccaacccac taagactact 420 tttaaataaaagaatgtttg tgggctatac acaaaactgg cagtgcaagc ttctgctaag 480 aatgaatgtaattttgggct aaaacaaagg tctctttttc ttggtaacct gtgtttttct 540 cacttccagtcacttgaaat tataattacc ttcttgaaac aaagaaatgg taatttattt 600 t 601 49 601DNA Homo sapiens 49 cacagccatc ctcataatac acaagcgcca ggagaggccaaagaaccttt actccaggac 60 acaaatgtgt gacgactgaa atcaggaaga tttttctatcagcacccagg tcttagtttt 120 cacctctagt tctggatgta cattccattt ccatccacagtgtactttaa gattgtctta 180 agaaatgtat ctgcatgaac tccgtgggaa ctaaaggaagtgggaactta gaaccagaca 240 gttttccaaa gatgttacaa tttcttttga aaaaccttttgtttattagc accaatttct 300 dgccactaag ctatttgttt tattatacat cctttaattaaaaactatat atgtaacttc 360 ttagatatta gcaaatgtct ctgctaccat ttccttaaggtgttgagctt taactctatg 420 ctgactcagt gagacacagt aggtagtatg gttgtggacctatttgtttt aacattgtaa 480 aattttgagt cagattttaa tattgtaaaa tcttgggtcaaataattcaa agccttaatg 540 cagatgcact aaaacaaaga aatggtaaat gaattgtttgcatttaaaaa aaaaaactct 600 t 601

That which is claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence selected from the group consisting of: SEQ ID NO:3 and SEQ IDNO:4; (b) an amino acid sequence of an allelic variant of an amino acidsequence selected from the group consisting of: SEQ ID NO:3 and SEQ IDNO:4, wherein said allelic variant is encoded by a nucleic acid moleculethat hybridizes under stringent conditions to the opposite strand of anucleic acid molecule selected from the group consisting of: SEQ IDNO:1, SEQ ID NO:2 and SEQ ID NO:5; (c) an amino acid sequence of anortholog of an amino acid sequence selected from the group consistingof: SEQ ID NO:3 and SEQ ID NO:4, wherein said ortholog is encoded by anucleic acid molecule that hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule selected from the groupconsisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5; and (d) afragment of an amino acid sequence selected from the group consistingof: SEQ ID NO:3 and SEQ ID NO:4, wherein said fragment comprises atleast 10 contiguous amino acids.
 2. An isolated peptide comprising anamino acid sequence selected from the group consisting of: (a) an aminoacid sequence selected from the group consisting of: SEQ ID NO:3 and SEQID NO:4; (b) an amino acid sequence of an allelic variant of an aminoacid sequence selected from the group consisting of: SEQ ID NO:3 and SEQID NO:4, wherein said allelic variant is encoded by a nucleic acidmolecule that hybridizes under stringent conditions to the oppositestrand of a nucleic acid molecule selected from the group consisting of:SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5; (c) an amino acid sequence ofan ortholog of an amino acid sequence selected from the group consistingof: SEQ ID NO:3 and SEQ ID NO:4, wherein said ortholog is encoded by anucleic acid molecule that hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule selected from the groupconsisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5; and (d) afragment of an amino acid sequence selected from the group consistingof: SEQ ID NO:3 and SEQ ID NO:4, wherein said fragment comprises atleast 10 contiguous amino acids.
 3. An isolated antibody thatselectively binds to a peptide of claim
 2. 4. An isolated nucleic acidmolecule consisting of a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence that encodes an amino acidsequence selected from the group consisting of: SEQ ID NO:3 and SEQ IDNO:4; (b) a nucleotide sequence that encodes of an allelic variant of anamino acid sequence selected from the group consisting of: SEQ ID NO:3and SEQ ID NO:4, wherein said nucleotide sequence hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeselected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQID NO:5; (c) a nucleotide sequence that encodes an ortholog of an aminoacid sequence selected from the group consisting of: SEQ ID NO:3 and SEQID NO:4, wherein said nucleotide sequence hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule selectedfrom the group consisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5;(d) a nucleotide sequence that encodes a fragment of an amino acidsequence selected from the group consisting of: SEQ ID NO:3 and SEQ IDNO:4, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 5. An isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence that encodes an amino acid sequence selectedfrom the group consisting of: SEQ ID NO:3 and SEQ ID NO:4; (b) anucleotide sequence that encodes of an allelic variant of an amino acidsequence selected from the group consisting of: SEQ ID NO:3 and SEQ IDNO:4, wherein said nucleotide sequence hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule selectedfrom the group consisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5;(c) a nucleotide sequence that encodes an ortholog of an amino acidsequence selected from the group consisting of: SEQ ID NO:3 and SEQ IDNO:4, wherein said nucleotide sequence hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule selectedfrom the group consisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5;(d) a nucleotide sequence that encodes a fragment of an amino acidsequence selected from the group consisting of: SEQ ID NO:3 and SEQ IDNO:4, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acidmolecule of claim
 5. 7. A transgenic non-human animal comprising anucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising anucleic acid molecule of claim
 5. 9. A host cell containing the vectorof claim
 8. 10. A method for producing any of the peptides of claim 1comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 11. A method for producing any of the peptides of claim 2comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 12. A method for detecting the presence of any of the peptidesof claim 2 in a sample, said method comprising contacting said samplewith a detection agent that specifically allows detection of thepresence of the peptide in the sample and then detecting the presence ofthe peptide.
 13. A method for detecting the presence of a nucleic acidmolecule of claim 5 in a sample, said method comprising contacting thesample with an oligonucleotide that hybridizes to said nucleic acidmolecule under stringent conditions and determining whether theoligonucleotide binds to said nucleic acid molecule in the sample.
 14. Amethod for identifying a modulator of a peptide of claim 2, said methodcomprising contacting said peptide with an agent and determining if saidagent has modulated the function or activity of said peptide.
 15. Themethod of claim 14, wherein said agent is administered to a host cellcomprising an expression vector that expresses said peptide.
 16. Amethod for identifying an agent that binds to any of the peptides ofclaim 2, said method comprising contacting the peptide with an agent andassaying the contacted mixture to determine whether a complex is formedwith the agent bound to the peptide.
 17. A pharmaceutical compositioncomprising an agent identified by the method of claim 16 and apharmaceutically acceptable carrier therefor.
 18. A method for treatinga disease or condition mediated by a human transporter protein, saidmethod comprising administering to a patient a pharmaceuticallyeffective amount of an agent identified by the method of claim
 16. 19. Amethod for identifying a modulator of the expression of a peptide ofclaim 2, said method comprising contacting a cell expressing saidpeptide with an agent, and determining if said agent has modulated theexpression of said peptide.
 20. An isolated human transporter peptidehaving an amino acid sequence that shares at least 70% homology with anamino acid sequence selected from the group consisting of: SEQ ID NO:3and SEQ ID NO:4.
 21. A peptide according to claim 20 that shares atleast 90 percent homology with an amino acid sequence selected from thegroup consisting of: SEQ ID NO:3 and SEQ ID NO:4.
 22. An isolatednucleic acid molecule encoding a human transporter peptide, said nucleicacid molecule sharing at least 80 percent homology with a nucleic acidmolecule selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2and SEQ ID NO:5.
 23. A nucleic acid molecule according to claim 22 thatshares at least 90 percent homology with a nucleic acid moleculeselected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQID NO:5.